Fluorescence polarization assays for determining clostridial toxin activity

ABSTRACT

The present invention provides a method of determining the presence or activity of a clostridial toxin by (a) treating with a sample, under conditions suitable for clostridial toxin protease activity, a clostridial toxin substrate which includes a fluorophore; a bulking group; and a clostridial toxin recognition sequence containing a cleavage site that intervenes between the fluorophore and the bulking group; (b) exciting the fluorophore with plane polarized light; and (c) determining fluorescence polarization of the treated substrate relative to a control substrate, where a change in fluorescence polarization of the treated substrate as compared to fluorescence polarization of the control substrate is indicative of the presence or activity of the clostridial toxin.

This application is a divisional and claims priority pursuant to 35U.S.C. §120 to U.S. patent application Ser. No. 10/948,097, filed Sep.22, 2004, which is hereby incorporated by reference in its entirety.

The present invention relates generally to protease assays, and morespecifically, to methods for determining the presence or activity ofclostridial toxins such as botulinum toxins and tetanus toxins usingfluorescence polarization.

The neuroparalytic syndrome of tetanus and the rare but potentiallyfatal disease, botulism, are caused by neurotoxins produced by bacteriaof the genus Clostridium. These clostridial neurotoxins are highlypotent and specific poisons of neural cells, with the human lethal doseof the botulinum toxins on the order of nanograms. Thus, the presence ofeven minute levels of botulinum toxins in foodstuffs represents a publichealth hazard that must be avoided through rigorous testing.

However, in spite of their potentially deleterious effects, lowcontrolled doses of botulinum neurotoxins have been successfully used astherapeutics and for some cosmetic applications. In particular,botulinum toxins have been used in the therapeutic management of avariety of focal and segmental dystonias, strabismus, and otherconditions in which a reversible depression of cholinergic nerveterminal activity is desired. Established therapeutic uses of botulinumneurotoxins in humans include, without limitation, treatment ofblepharospasm, hemifacial spasm, laringeal dysphonia, focalhyperhidrosis, hypersalivation, oromandibular dystonia, cervicaldystonia, torticollis, strabismus, limbs dystonia, occupational crampsand myokymia (Rossetto et al., Toxicon 39:27-41 (2001)). As an example,intramuscular injection of spastic tissue with small quantities ofbotulinum neurotoxin A has been used effectively to treat spasticity dueto brain injury, spinal cord injury, stroke, multiple sclerosis andcerebral palsy. Additional possible clinical uses of clostridialneurotoxins are currently being investigated.

Given the potential danger associated with small quantities of botulinumtoxins in foodstuffs and the need to prepare accurate pharmaceuticalformulations, assays for botulinum neurotoxins presently are employed inthe food and pharmaceutical industries. The food industry requiresassays for the botulinum neurotoxins to validate new food packagingmethods and to ensure food safety. The growing clinical use of thebotulinum toxins necessitates accurate assays for botulinum neurotoxinactivity for product formulation as well as quality control. In bothindustries, a mouse lethality test currently is the only acceptableassay for botulinum neurotoxin potency.

Unfortunately, the mouse lethality assay suffers from several drawbacks:cost due to the large numbers of laboratory animals required; lack ofspecificity; potential for inaccuracy unless large animal groups areused; and sacrifice of animal life. Thus, there is a need for newmethods based on convenient synthetic substrates that can complement andreduce the need for the mouse lethality assay. The present inventionsatisfies this need by providing novel assays for determining thepresence or activity of a clostridial toxin and provides relatedadvantages as well.

The present invention provides a method of determining the presence oractivity of a clostridial toxin by (a) treating with a sample, underconditions suitable for clostridial toxin protease activity, aclostridial toxin substrate which includes a fluorophore; a bulkinggroup; and a clostridial toxin recognition sequence containing acleavage site that intervenes between the fluorophore and the bulkinggroup; (b) exciting the fluorophore with plane polarized light; and (c)determining fluorescence polarization of the treated substrate relativeto a control substrate, where a change in fluorescence polarization ofthe treated substrate as compared to fluorescence polarization of thecontrol substrate is indicative of the presence or activity of theclostridial toxin.

Further provided herein is a method of determining the presence oractivity of a clostridial toxin by (a) treating with a sample, underconditions suitable for clostridial toxin protease activity, aclostridial toxin substrate containing (i) a donor fluorophore; (ii) anacceptor having an absorbance spectrum overlapping the emission spectrumof the donor fluorophore; and (iii) a clostridial toxin recognitionsequence containing a cleavage site, where the cleavage site intervenesbetween the donor fluorophore and the acceptor and where, under theappropriate conditions, resonance energy transfer is exhibited betweenthe donor fluorophore and the acceptor; (b) exciting the donorfluorophore with plane polarized light; and (c) determining fluorescencepolarization of the treated substrate relative to a control substrate,where a change in fluorescence polarization of the treated substrate ascompared to fluorescence polarization of the control substrate isindicative of the presence or activity of the clostridial toxin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the four steps required for tetanus andbotulinum toxin activity in central and peripheral neurons.

FIG. 2 shows the subcellular localization and sites of cleavage ofSNAP-25, VAMP and syntaxin. VAMP is bound to synaptic vesicle membrane,whereas SNAP-25 and syntaxin are bound to the target plasma membrane.BoNT/A and /E cleave SNAP-25 close to the carboxy-terminus, releasingnine or 26 residues, respectively. BoNT/B, /D, /F, /G and TeNT act onthe conserved central portion of VAMP (dotted) and release theamino-terminal portion of VAMP into the cytosol. BoNT/C1 cleaves SNAP-25close to the carboxy-terminus as well as cleaving syntaxin at a singlesite near the cytosolic membrane surface. The action of BoNT/B, /C1, /D,/F, /G and TeNT results in release of a large portion of the cytosolicdomain of VAMP or syntaxin, while only a small portion of SNAP-25 isreleased by selective proteolysis of BoNT/A, /C1 or /E.

FIG. 3 shows an alignment of various SNAP-25 proteins. Human SNAP-25(SEQ ID NO: 1; GenBank accession g4507099; see, also, related humanSNAP-25 sequence g2135800); mouse SNAP-25 (SEQ ID NO: 2; GenBankaccession G6755588); Drosophila SNAP-25 (SEQ ID NO: 3; GenBank accessiong548941); goldfish SNAP-25 (SEQ ID NO: 4; GenBank accession g2133923);sea urchin SNAP-25 (SEQ ID NO: 5; GenBank accession g2707818) andchicken SNAP-25 (SEQ ID NO: 6; GenBank accession g481202) are depicted.

FIG. 4 shows an alignment of various VAMP proteins. Human VAMP-1 (SEQ IDNO: 7; GenBank accession g135093); human VAMP-2 (SEQ ID NO: 8; GenBankaccession g135094); mouse VAMP-2 (SEQ ID NO: 9; GenBank accessiong2501081); bovine VAMP (SEQ ID NO: 10; GenBank accession g89782); frogVAMP (SEQ ID NO: 11; GenBank accession g6094391); and sea urchin VAMP(SEQ ID NO: 12; GenBank accession g5031415) are depicted.

FIG. 5 shows an alignment of various syntaxin proteins. Human syntaxin1A (SEQ ID NO: 13; GenBank accession g15079184), human syntaxin 1B2 (SEQID NO: 14; GenBank accession g15072437), mouse syntaxin 1A (SEQ ID NO:15; GenBank accession g15011853), Drosophila syntaxin 1A (SEQ ID NO: 16;GenBank accession g2501095); C. elegans syntaxin A (SEQ ID NO: 17;GenBank accession g7511662) and sea urchin syntaxin (SEQ ID NO: 18;GenBank accession g13310402) are depicted.

FIG. 6 shows (A) a schematic of plasmid pQBI GFP-SNAP25₍₁₃₄₋₂₀₆₎₋6×HIS-Cand (B) the nucleic acid and amino acid sequences (SEQ ID NOS: 19 and20) of pQBI GFP-SNAP25₍₁₃₄₋₂₀₆₎₋6×HIS-C.

FIG. 7 shows (A) the absorption spectrum and (B) the excitation (dotted)and emission (bold) spectra of GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6C.

FIG. 8 shows (A) the UV-VIS absorption spectrum and (B) the excitation(bold) and emission (dotted) spectra of GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6C-AlexaFluor® 594.

FIG. 9 shows turnover of the GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6C-Alexa Fluor® 594substrate using reduced BoNT/A at various concentrations. The arrowindicates when the reduced toxin complex was added.

FIG. 10 shows turnover of the GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6C-Alexa Fluor® 546substrate using recombinant BoNT/A light chain. The arrow indicatesaddition of the BoNT/A light chain.

DETAILED DESCRIPTION

The invention provides novel methods for determining the presence oractivity of clostridial toxins including botulinum toxins of allserotypes as well as tetanus toxins. The novel methods of the invention,which rely on a clostridial toxin substrate useful for fluorescencepolarization analysis, reduce the need for animal toxicity studies andcan be used to analyze crude and bulk samples as well as highly purifieddichain or single chain toxins or formulated toxin products. Thefluorescence polarization-based methods of the invention areadvantageous in that they are sensitive assays which are robust in termsof interference from background fluorescence present in samples.Furthermore, the novel methods of the invention can be performed ashomogeneous solution-phase assays and are amenable to automatedhigh-throughput formats.

As disclosed herein in Example I, a clostridial toxin substrate wasprepared with Alexa Fluor® 594 as a fluorophore, green fluorescentprotein (GFP) as a bulking group, and a portion of SNAP-25 (residues134-206) as a clostridial toxin recognition sequence for BoNT/A. Theabsorption spectrum of the GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-Cys protein labeledwith Alexa Fluor® 594 is shown herein in FIG. 8A, and the excitation andemission spectra of GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C-Alexa Fluor® 594 areshown herein in FIG. 8B. As further disclosed herein in Example II, theGFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C-Alexa Fluor® 594 substrate was tested for itsutility as a suitable substrate by assaying for the activity of BoNT/Areduced bulk toxin by recording the change in fluorescence polarizationover time. As shown in FIG. 9, there was a reduction in fluorescencepolarization at or shortly after the time the diluted bulk BoNT/A toxinwas added, and toxin activity was detected at a concentration of aslittle as about 50 ng/ml (see panel 9D). These results demonstrate thatthe presence or activity of clostridial toxins can be determined usingsynthetic substrates assayed by fluorescence polarization.

As further disclosed herein, fluorescence polarization can be combinedwith fluorescence resonance energy transfer to sensitively assay for thepresence or activity of a clostridial toxin. As disclosed in Example I,a GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C protein was site-specifically labeled atthe carboxy-terminal cysteine residue with Alexa Fluor® 546; thephotoselection properties of GFP and Alexa Fluor® 546 provide forfluorescence resonance energy transfer (FRET) between the donorfluorophore GFP and the acceptor Alexa Fluor® 546. As disclosed inExample III and shown in FIG. 10, fluorescence polarization increasedupon addition of recombinant BoNT/A light chain. Without wishing to bebound by the following, FRET in the intact substrate leads to anapparent depolarization of Alexa Fluor® 546 emission due to thesignificant angle between the initially selected dipole (GFP) and thedipole which would be selected by direct excitation of Alexa Fluor® 546.Upon proteolysis, the FRET effect is abolished, and polarizationconsequently increases even though rotation of the Alexa Fluor® dye isincreased. The combination of fluorescence resonance energy transferwith fluorescence polarization enhanced the polarization change uponturnover, increasing the sensitivity of the assay (see FIG. 10). Theseresults indicate that fluorescence polarization can be combined withfluorescence resonance energy transfer for enhanced sensitivity inassaying for the presence or activity of a clostridial toxin.

Based on these findings, the present invention provides a method ofdetermining the presence or activity of a clostridial toxin by (a)treating with a sample, under conditions suitable for clostridial toxinprotease activity, a clostridial toxin substrate which includes afluorophore; a bulking group; and a clostridial toxin recognitionsequence containing a cleavage site that intervenes between thefluorophore and the bulking group; (b) exciting the fluorophore withplane polarized light; and (c) determining fluorescence polarization ofthe treated substrate relative to a control substrate, where a change influorescence polarization of the treated substrate as compared tofluorescence polarization of the control substrate is indicative of thepresence or activity of the clostridial toxin. In one embodiment, thechange in fluorescence polarization is a decrease in fluorescencepolarization. In another embodiment, step (c) includes determining thechange in fluorescence polarization of the treated substrate over time.

In a method of the invention, a fluorophore can have, withoutlimitation, a fluorescence lifetime of at least 0.5 nanoseconds, or atleast 5 nanoseconds, or at least 10 nanoseconds. Any of a variety offluorophores can be useful in the methods of the invention including,but not limited to, Alexa Fluor® dyes; fluorescein and fluoresceinderivatives such as diaminotriazinylamino-fluorescein (DTAF); biarsenicderivatives of fluorescein such as fluorescein arsenical hairpin bindingdye (FlAsH™) and red biarsenical dye (ReAsH™); carboxyfluorescein (FAM);Texas Red™; tetramethylcarboxyrhodamine (TMR); carboxy-x-rhodamine(ROX); rhodamine green; Oregon Green 488; BODIPY-TR®; BODIPY-TMR;BODIPY®-FL; Cy3; Cy™3B and Dansyl. In one embodiment, the fluorophore isan Alexa Fluor® dye such as, without limitation, Alexa Fluor® 594. Inother embodiments, the fluorophore is FlAsH™ or ReAsH™.

A variety of bulking groups are useful in the methods of the invention,including, without limitation, fluorescent proteins such as greenfluorescent protein. In one embodiment, a method of the invention ispracticed such that the change in molecular mass upon cleavage of theclostridial toxin substrate is at least 1000 Da. In a furtherembodiment, a method of the invention is practiced such that thedecrease in fluorescence polarization is at least 5 millipolarizationunits (mP). In still a further embodiment, a method of the invention ispracticed such that the decrease in fluorescence polarization is atleast 15 mP.

A variety of recognition sequences can be included in a clostridialtoxin substrate useful in a method of the invention. In one embodiment,the recognition sequence is a BoNT/A recognition sequence such as,without limitation, a BoNT/A recognition sequence containing at leastsix consecutive residues of SNAP-25, where the six consecutive residuesinclude Gln-Arg, or a peptidomimetic thereof. Such a BoNT/A recognitionsequence can include, for example, residues 134 to 206 of SEQ ID NO: 2.A recognition sequence included in a clostridial toxin substrate usefulin a method of the invention also can be, without limitation, a BoNT/Brecognition sequence. Such a BoNT/B recognition sequence can contain,for example, at least six consecutive residues of VAMP, where the sixconsecutive residues include Gln-Phe, or a peptidomimetic thereof. In afurther embodiment, a recognition sequence included in a clostridialtoxin substrate useful in a method of the invention is a BoNT/C1recognition sequence. Such a BoNT/C1 recognition sequence can contain,without limitation, at least six consecutive residues of syntaxin, wherethe six consecutive residues include Lys-Ala, or a peptidomimeticthereof. A BoNT/C1 recognition sequence useful in the invention also cancontain at least six consecutive residues of SNAP-25, where the sixconsecutive residues include Arg-Ala, or a peptidomimetic thereof.

In a further embodiment, a recognition sequence included in aclostridial toxin substrate useful in a method of the invention is aBoNT/D recognition sequence. Such a BoNT/D recognition sequence cancontain, for example, at least six consecutive residues of VAMP, wherethe six consecutive residues include Lys-Leu, or a peptidomimeticthereof. A recognition sequence useful in the invention also can be, forexample, a BoNT/E recognition sequence. Such a BoNT/E recognitionsequence can include, without limitation, residues 134 to 206 of SEQ IDNO: 2, or can contain at least six consecutive residues of SNAP-25,where the six consecutive residues include Arg-Ile, or a peptidomimeticthereof. In yet another embodiment, a recognition sequence included in aclostridial toxin substrate useful in a method of the invention is aBoNT/F recognition sequence. BoNT/F recognition sequences useful in theinvention encompass, without limitation, those having at least sixconsecutive residues of VAMP, where the six consecutive residues includeGln-Lys, or a peptidomimetic thereof. A recognition sequence included ina clostridial toxin substrate useful in a method of the invention alsocan be a BoNT/G recognition sequence. Such BoNT/G recognition sequencesencompass, without limitation, those having at least six consecutiveresidues of VAMP, where the six consecutive residues include Ala-Ala, ora peptidomimetic thereof. In still a further embodiment, a recognitionsequence included in a clostridial toxin substrate useful in a method ofthe invention is a TeNT recognition sequence. Such a TeNT recognitionsequence can be, without limitation, a sequence containing at least sixconsecutive residues of VAMP, where the six consecutive residues includeGln-Phe, or a peptidomimetic thereof.

Any of a variety of clostridial toxin substrates are useful fordetermining the presence or activity of a clostridial toxin according toa method of the invention. In one embodiment, a clostridial toxinsubstrate is a peptide or peptidomimetic having at least 100 residues.In another embodiment, a clostridial toxin substrate is a peptide orpeptidomimetic having at least 200 residues. Furthermore, any of avariety of samples can be assayed according to a method of the inventionincluding, but not limited to, crude cell lysates, isolated clostridialtoxins including isolated clostridial toxin light chains; and formulatedclostridial toxin products such as, without limitation, formulatedBoNT/A, BoNT/B or BoNT/E toxin products.

The tetanus and botulinum neurotoxins which can be assayed according toa method of the invention are produced by Clostridia. These toxins causethe neuroparalytic syndromes of tetanus and botulism, with tetanus toxinacting mainly within the central nervous system and botulinum toxinacting on the peripheral nervous system. Clostridial neurotoxins share asimilar mechanism of cell intoxication in which the release ofneurotransmitters is blocked. In these toxins, which are composed of twodisulfide-linked polypeptide chains, the larger subunit is responsiblefor neurospecific binding and translocation of the smaller subunit intothe cytoplasm. Upon translocation and reduction in neurons, the smallerchain displays peptidase activity specific for protein componentsinvolved in neuroexocytosis. The “SNARE” protein targets of clostridialtoxins are common to exocytosis in a variety of non-neuronal types; inthese cells, as in neurons, light chain peptidase activity inhibitsexocytosis.

Tetanus neurotoxin and botulinum neurotoxins B, D, F, and G specificallyrecognize VAMP (also known as synaptobrevin), an integral protein of thesynaptic vesicle membrane. VAMP is cleaved at distinct bonds dependingon the neurotoxin. Botulinum A and E neurotoxins recognize and cleavespecifically SNAP-25, a protein of the presynaptic membrane, at twodifferent sites in the carboxy-terminal portion of the protein.Botulinum neurotoxin C cleaves syntaxin, a protein of the nerveplasmalemma, in addition to SNAP-25. The three protein targets of theClostridial neurotoxins are conserved from yeast to humans althoughcleavage sites and toxin susceptibility are not necessarily conserved(see below; see, also, Humeau et al., Biochimie 82:427-446 (2000);Niemann et al., Trends in Cell Biol. 4:179-185 (1994); and Pellizzari etal., Phil. Trans. R. Soc. London 354:259-268 (1999)).

Naturally occurring tetanus and botulinum neurotoxins are produced aspolypeptide chains of 150 kDa without a leader sequence. These toxinsmay be cleaved by bacterial or tissue proteinases at an exposedprotease-sensitive loop, generating active dichain toxin. Selectiveproteolytic cleavage activates the toxins by generating twodisulfide-linked chains: an L chain of 50 kDa and an H chain of 100 kDa,which is composed of two domains denoted H_(N) and H_(C). This dichaintoxin is more active than unnicked toxin. Naturally occurringclostridial toxins contain a single interchain disulfide bond bridgingthe heavy chain and light chain; such a bridge is important forneurotoxicity of toxin added extracellularly (Montecucco and Schiavo,Quarterly Rev. Biophysics 28:423-472 (1995)).

The clostridial toxins appear to be folded into three distinct domainsof about 50 kDa which are connected by loops, with each domain having adistinct functional role. As illustrated in FIG. 1, the cellintoxication mechanism of the clostridial toxins consists of fourdistinct steps: (1) binding; (2) internalization; (3) membranetranslocation; and (4) enzymatic target modification. Thecarboxy-terminal domain of the heavy chain (H_(C)) functions inneurospecific binding, while the amino-terminal domain of the H chain(H_(N)) functions in membrane translocation from endosome to cellcytoplasm. Following reduction of the disulfide linkage inside the cell,the zinc-endopeptidase activity of the L chain is liberated (Montecuccoand Schiavo, supra, 1995).

The amino acid sequences of eight human clostridial neurotoxin serotypeshave been derived from the corresponding genes (Niemann, “MolecularBiology of Clostridial Neurotoxins” in Sourcebook of Bacterial ProteinToxins Alouf and Freer (Eds.) pp. 303-348 London: Academic Press 1991).The L chain and H chain are composed of roughly 439 and 843 residues,respectively. Homologous segments are separated by regions of little orno similarity. The most well conserved regions of the L chain are theamino-terminal portion (100 residues) and central region (correspondingto residues 216 to 244 of TeNT), as well as the two cysteines formingthe interchain disulfide bond. The 216 to 244 region contains aHis-Glu-X-X-His binding motif characteristic of zinc-endopeptidases.

The clostridial toxin heavy chains are less well conserved than thelight chains, with the carboxy-terminal portion of H_(C) correspondingto residues 1140 to 1315 of TeNT the most variable. This is consistentwith the involvement of the H_(C) domain in binding to nerve terminalsand the fact that different neurotoxins appear to bind differentreceptors.

Comparison of the nucleotide and amino acid sequences of the clostridialtoxins indicates that they derive from a common ancestral gene.Spreading of these genes may have been facilitated by the fact that theclostridial neurotoxin genes are located on mobile genetic elements. Asdiscussed further below, sequence variants of the seven botulinum toxinsare known in the art. See, for example, Humeau et al., supra, 2000.

As discussed above, natural targets of the clostridial neurotoxinsinclude VAMP, SNAP-25, and syntaxin. VAMP is associated with thesynaptic vesicle membrane, whereas SNAP-25 and syntaxin are associatedwith the target membrane (see FIG. 2). BoNT/A and BoNT/E cleave SNAP-25in the carboxy-terminal region, releasing nine or twenty-six amino acidresidues, respectively, and BoNT/C1 also cleaves SNAP-25 near thecarboxy-terminus. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F andBoNT/G, and tetanus toxin, act on the conserved central portion of VAMP,and release the amino-terminal portion of VAMP into the cytosol. BoNT/C1cleaves syntaxin at a single site near the cytosolic membrane surface.Thus, BoNT/B, BoNT/C1, BoNT/D, BoNT/F, BoNT/G or TeNT proteolysisresults in release of a large portion of the cytosolic domain of VAMP orsyntaxin, while only a small portion of SNAP-25 is released by BoNT/A,BoNT/C1 or BoNT/E cleavage (Montecucco and Schiavo, supra, 1995).

Naturally occurring SNAP-25, a protein of about 206 residues lacking atransmembrane segment, is associated with the cytosolic surface of thenerve plasmalemma (FIG. 2; see, also, Hodel et al., Int. J. Biochemistryand Cell Biology 30:1069-1073 (1998)). In addition to homologs highlyconserved from Drosophila to mammals, SNAP-25-related proteins also havebeen cloned from yeast. SNAP-25 is required for axonal growth duringdevelopment and may be required for nerve terminal plasticity in themature nervous system. In humans, two isoforms are differentiallyexpressed during development; isoform a is constitutively expressedduring fetal development, while isoform b appears at birth andpredominates in adult life. SNAP-25 analogues such as SNAP-23 also areexpressed outside the nervous system, for example, in pancreatic cells.

Naturally occurring VAMP is a protein of about 120 residues, with theexact length depending on the species and isotype. As shown in FIG. 2,VAMP contains a short carboxy-terminal segment inside the vesicle lumenwhile most of the molecule is exposed to the cytosol. The proline-richamino-terminal thirty residues are divergent among species and isoformswhile the central portion of VAMP (residues 30 to 96), which is rich incharged and hydrophilic residues and includes known cleavage sites, ishighly conserved. VAMP colocalizes with synaptophysin on synapticvesicle membranes.

A variety of species homologs of VAMP are known in the art includinghuman, rat, bovine, Torpedo, Drosophila, yeast, squid and Aplysiahomologs. In addition, multiple isoforms of VAMP have been identifiedincluding VAMP-1, VAMP-2 and cellubrevin, and forms insensitive to toxincleavage have been identified in non-neuronal cells. VAMP appears to bepresent in all vertebrate tissues although the distribution of VAMP-1and VAMP-2 varies in different cell types. Chicken and rat VAMP-1 arenot cleaved by TeNT or BoNT/B. These VAMP-1 homologs have a valine inplace of the glutamine present in human and mouse VAMP-1 at the TeNT orBoNT/B cleavage site. The substitution does not affect BoNT/D, /F or /G,which cleave both VAMP-1 and VAMP-2 with similar rates.

Syntaxin is located on the cytosolic surface of the nerve plasmalemmaand is membrane-anchored via a carboxy-terminal segment, with most ofthe protein exposed to the cytosol. Syntaxin colocalizes with calciumchannels at the active zones of the presynaptic membrane, whereneurotransmitter release takes place. In addition, syntaxin interactswith synaptotagmin, a protein of the SSV membrane, that forms afunctional bridge between the plasmalemma and the vesicles. A variety ofsyntaxin isoforms have been identified. Two isoforms of slightlydifferent length (285 and 288 residues) have been identified in nervecells (isoforms 1A and 1B), with isoforms 2, 3, 4 and 5 expressed inother tissues. The different isoforms have varying sensitivities toBoNT/C1, with the 1A, 1B, 2 and 3 syntaxin isoforms cleaved by thistoxin.

The methods of the invention rely, in part, on the use of fluorescencepolarization. According to the theory of fluorescence polarization, whena fluorescently labeled molecule is excited with plane polarized light,it emits lights that has a degree of polarization which is inverselyproportional to its molecular rotation. As a consequence, for largefluorescently labeled molecules, which remain relatively stationaryduring their excited state (about 4 ns for fluorescein), polarizationremains relatively constant between excitation and emission. Incontrast, small fluorescently labeled molecules rotate rapidly duringthe excited state, such that polarization of the light changessignificantly between excitation and emission. Therefore, as ageneralization, small molecules have low polarization values, and largemolecules have high polarization values. See, for example, Weber,“Polarization of the Fluorescence of Solutions” in Fluorescence &Phosphorescence Analysis pages 217-241 Wiley Interscience (1996), andJameson and Seifried, Methods Enzym. 19:222-233 (1999).

Fluorescence polarization assays are homogeneous in that they do notrequire a separation step and do not require attachment of substrate toan immobilized phase. Furthermore, polarization values can be measuredrepeatedly. In addition, fluorescence polarization is a sensitivetechnique which can be used to measure polarization values offluorophores from low picomolar and micromolar levels. Polarization isalso independent of fluorescence intensity.

Fluorescence anisotropy (commonly denoted as “r” or sometimes “A”) is analternative definition of how a plane of polarized light changes betweenexcitation and emission with a rotating fluorophore. Fluorescencepolarization and anisotropy are well known in the art as described inLundblad et al., Mol. Endocrin. 10:607-612 (1996); Nasir et al., Comb.Chem. High Throughput Screen. 2:177-190 (1999); Sittampalam et al.,Curr. Opin. Chem. Biol. 1:384-391 (1997); Thompson et al., Biotechniques32:34-40 (1997); Lakowicz et al., J. Biomol. Screen. 5:123-132 (2000);and Fernandes, Curr. Opin. Chem. Biol. 2:597-603 (1998).

In particular, fluorescence polarization (P) and anisotropy (r) aredefined as follows:

${Polarization} = {P = \frac{I_{Vertical} - I_{Horizontal}}{I_{Vertical} + I_{Horizontal}}}$and${Anisotrophy} = {r = \frac{I_{Vertical} - I_{Horizontal}}{I_{Vertical} + {2*I_{Horizontal}}}}$

where I_(Vertical) is the intensity of the emission light parallel tothe excitation light plane and I_(Horizontal) is the intensity of theemission light perpendicular to the excitation light plane. P and r,being ratios of light intensities, are dimensionless. Experimental datacan be expressed in millipolarization units, where 1 polarizationunit=1000 mP units, or in millianisotropy units, where 1 anisotropyunit=1000 mA units.

The formulae to interconvert polarization and anisotropy are as follows:

$P = \frac{3r}{( {2 + r} )}$ and$r = \frac{2P}{( {3 - P} )}$

Fundamentally, polarization is a relationship of fluorescence lifetimeand how fast a fluorophore rotates in the time between excitation andemission. The principal factors controlling rotation are molar volume(V), absolute temperature (T), and viscosity (η). The rotationalcorrelation time (Θ) and the rotational relaxation time (ρ_(o)) aretaken from the work of Perrin and Weber. In particular, the rotationalcorrelation time (Θ) is taken from the Perrin equation as follows:

$( {\frac{1}{P} - \frac{1}{3}} ) = {( {\frac{1}{P_{o}} - \frac{1}{3}} )*( {1 + {\tau/ \ominus}} )}$

and is defined as:

Rotational Correlation Time

$( \ominus ) = \frac{\eta \; V}{RT}$

Furthermore, the rotational relaxation time (ρ_(o)) is taken from thePerrin/Weber equation (Perrin, J. Phys. Rad. 7:390-401 (1926)), asfollows:

$( {\frac{1}{P} - \frac{1}{3}} ) = {( {\frac{1}{P_{o}} - \frac{1}{3}} )*( {1 + {3{\tau/P}}} )}$

and is defined as:

Rotational Relaxation Time

$( \rho_{o} ) = \frac{3\eta \; V}{RT}$

-   -   where R is the gas constant, τ is the fluorescence lifetime, P        is the polarization, and P₀ is the limiting polarization.

From the above, it can be seen that, where lifetime, viscosity, andtemperature are held constant, the molecular volume (and thus thepolarization or anisotropy) determines the rotation. The larger themolecular volume, the slower the molecule rotates and the higher thepolarization and anisotropy values. Furthermore, as is evident from theequations above, the rotational relaxation time will be exactly threetimes longer than the rotational correlation time.

A method of the invention relies on a clostridial toxin substrate whichincludes, in part, a fluorophore. As used herein, the term “fluorophore”means a molecule that, when irradiated with light of a certainwavelength, emits light, also denoted fluorescence, of a differentwavelength. The term fluorophore is synonymous in the art with the term“fluorochrome.”

Fluorophores useful in the invention, as well as donor fluorophoreswhich are discussed further below, include those having fluorescencelifetimes suitable for fluorescence polarization analysis. Usefulfluorophores include, without limitation, Alexa Fluor® dyes; fluoresceinand fluorescein derivatives such as diaminotriazinylamino-fluorescein(DTAF); biarsenic derivatives of fluorescein such as fluoresceinarsenical hairpin binding dye (FlAsH™) and red biarsenical dye (ReAsH™);carboxyfluorescein (FAM); Texas Red™; tetramethylcarboxyrhodamine (TMR);carboxy-x-rhodamine (ROX); rhodamine green; Oregon Green 488;BODIPY-TR®; BODIPY-TMR; BODIPY®-FL; Cy3, Cy™3B and Dansyl. Additionalfluorophores suitable for fluorescence polarization are known in theart, including, but not limited to, long-wavelength fluorophores such asBODIPY-TMR and BODIPY-TR® (Molecular Probes), which tend to minimizeassay interference, and pH insensitive fluorophores such as BODIPY-FL.See, for example, Owicki, J. Biomol. Screening 5:297-306 (2000); Burkeet al., Comb. Chem. & High Throughput Screen. 6:183-194 (2003); andJameson and Croney, Comb. Chem. & High Throughput Screen. 6:167-176(2003). A variety of fluorophores and donor fluorophores useful forfluorescence polarization are commercially available from varioussources such as Molecular Probes (Eugene, Oreg.) and Amersham PharmaciaBiotech (Piscataway, N.J.). One skilled in the art understands thatthese as well as other fluorophores suitable for fluorescencepolarization are known in the art and can be useful in the methods ofthe invention.

As used herein, the term “bulking group” means a moiety havingsufficient hydrodynamic volume such that, upon cleavage of a clostridialtoxin substrate into which the bulking group is incorporated, there is achange in polarization of at least 3 millipolarization units (mP).

Any of a variety of moieties can be useful as a bulking group in amethod of the invention including physical, chemical and biologicalmoieties which can be covalently or non-covalently incorporated into aclostridial toxin substrate. In one embodiment, the bulking group isexpressed as a fusion protein with another component of the clostridialtoxin substrate. Bulking groups useful in the invention encompassnatural and man-made moieties and further encompass, without limitation,inert moieties as well as those with biological or other activity. Abulking group useful in the invention can be, without limitation, amoiety having a size of greater than 1000 Da. A bulking group useful inthe invention also can be, without limitation, a moiety having a size ofgreater than 2 kDa, 3 kDa, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa,30 kDa, 35 kDa or 40 kDa. See, also, Mattison et al., Application Notefor Protein Solutions Inc. February 2001. One skilled in the artunderstands that a fluorophore with a suitable lifetime will be selecteddepending, in part, on the size of the bulking group.

A variety of bulking groups can be useful in the invention. Asnon-limiting examples, a bulking group useful in the invention can be aninert or active protein, peptide or peptidomimetic; an antibody; organicchemical; latex or other bead; or moiety such as streptavidin.Additional bulking groups useful in the invention encompass, withoutlimitation, phage and other viruses; cells; liposomes; polymeric andnon-polymeric matrices; gold and other particles; and microdevices andnanodevices. As non-limiting examples, a bulking group useful in theinvention can be a fluorescent protein such as GFP or BFP, or a fragmentthereof; a protein useful for affinity purification such asglutathione-S-transferase (GST) or maltose-binding protein (MBP); or anantibody such as, without limitation, an anti-FLAG, anti-hemagluttinin(HA) or anti-myc antibody. Streptavidin also can be a bulking groupuseful in the invention. As a non-limiting example, a biotinylationsequence can be covalently included in a clostridial toxin substrate,providing for association with streptavidin; enzymatic cleavage can bedetected by following the fluorescence polarization change upon additionof streptavidin as described in Levine et al., “Measurement of specificprotease activity utilizing fluorescence polarization,” Anal. Biochem.247:83-88 (1997).

A clostridial toxin substrate useful in the invention contains acleavage site that “intervenes” between a fluorophore and a bulkinggroup. Thus, the cleavage site is positioned in between the fluorophoreand the bulking group such that proteolysis at the cleavage site resultsin a first cleavage product containing the fluorophore and a secondcleavage product containing the bulking group. It is understood that allor only a portion of the clostridial toxin recognition sequence mayintervene between the fluorophore and the bulking group.

A clostridial toxin substrate useful in the invention contains, in part,a clostridial toxin recognition sequence which includes a cleavage site.By definition, a clostridial toxin substrate is susceptible to cleavageby at least one clostridial toxin under conditions suitable forclostridial toxin protease activity.

As used herein, the term “clostridial toxin recognition sequence” meansa scissile bond together with adjacent or non-adjacent recognitionelements, or both, sufficient for detectable proteolysis at the scissilebond by a clostridial toxin under conditions suitable for clostridialtoxin protease activity. A variety of clostridial toxin recognitionsequences are discussed herein below.

In particular embodiments, a clostridial toxin substrate useful in theinvention is a peptide or peptidomimetic having a defined length. Aclostridial toxin substrate can be, for example, a peptide orpeptidomimetic having at least 50, at least 100, at least 150, at least200, at least 250, at least 300, at least 350, at least 500, at least600, at least 700, at least 800 or at least 900 residues. In otherembodiments, a clostridial toxin substrate has at most 20 residues, atmost 30 residues, at most 40 residues, at most 50 residues, at most 60residues, at most 70 residues, at most 80 residues, at most 90 residues,at most 100 residues, at most 150 residues, at most 200 residues, atmost 250 residues, at most 300 residues, at most 350 residues or at most400 residues.

It is understood that a clostridial toxin substrate useful in theinvention optionally can include one or more additional components. As anon-limiting example, a flexible spacer sequence such as GGGGS (SEQ IDNO: 21) can be included in a clostridial toxin substrate useful in theinvention. A useful clostridial toxin substrate further can include,without limitation, one or more of the following: an affinity tag suchas HIS6; biotin or a biotinylation sequence; or an epitope such as FLAG,hemagluttinin (HA), c-myc, or AU1; an immunoglobulin hinge region; anN-hydroxysuccinimide linker; a peptide or peptidomimetic hairpin turn;or a hydrophilic sequence or another component or sequence that, forexample, facilitates purification or promotes the solubility orstability of the clostridial toxin substrate.

As discussed further below, it is understood that the methods of theinvention are applicable to crude samples as well as highly purifieddichain and single chain toxins. As non-limiting examples, a method ofthe invention can be useful to determine the presence or activity of aclostridial toxin in a food or beverage sample; to assay a sample from ahuman or animal, for example, exposed to a clostridial toxin or havingone or more symptoms of a clostridial toxin; to follow activity duringproduction and purification of clostridial toxin; or to assay formulatedclostridial toxin products such as pharmaceuticals or cosmetics.

A variety of samples are useful in the methods of the invention. As usedherein, the term “sample” means any biological matter that contains orpotentially contains an active clostridial toxin. Thus, the term sampleencompasses, but is not limited to, purified or partially purifiedclostridial toxin; recombinant single chain or dichain toxin with anaturally or non-naturally occurring sequence; recombinant clostridialtoxin with a modified protease specificity; recombinant clostridialtoxin with an altered cell specificity; chimeric toxin containingstructural elements from multiple clostridial toxin species or subtypes;bulk toxin; formulated toxin product; cells or crude, fractionated orpartially purified cell lysates, for example, engineered to include arecombinant nucleic acid encoding a clostridial toxin; bacterial,baculoviral and yeast lysates; raw, cooked, partially cooked orprocessed foods; beverages; animal feed; soil samples; water samples;pond sediments; lotions; cosmetics; and clinical formulations. Itfurther is understood that the term sample encompasses tissue samples,including, without limitation, mammalian tissue samples, livestocktissue samples such as sheep, cow and pig tissue samples; primate tissuesamples; and human tissue samples. Such samples encompass, withoutlimitation, intestinal samples such as infant intestinal samples, andtissue samples obtained from a wound.

As discussed further below, a variety of conditions suitable forclostridial toxin protease activity are useful in the methods of theinvention. For example, conditions suitable for clostridial toxinprotease activity can be provided such that at least 10% of thesubstrate is cleaved. Similarly, conditions suitable for clostridialtoxin protease activity can be provided such that at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or 95% of the clostridial toxin substrateis cleaved, or such that 100% of the clostridial toxin substrate iscleaved. In one embodiment, the conditions suitable for clostridialtoxin protease activity are selected such that the assay is linear. Inanother embodiment, conditions suitable for clostridial toxin proteaseactivity are provided such that at least 90% of the clostridial toxinsubstrate is cleaved. In a further embodiment, conditions suitable forclostridial toxin protease activity are provided such that at most 25%of the clostridial toxin substrate is cleaved. In yet furtherembodiments, conditions suitable for clostridial toxin protease activityare provided such that at most 5%, 10%, 15% or 20% of the clostridialtoxin substrate is cleaved.

In the methods of the invention, the clostridial toxin substrate can betreated with a sample in solution phase. As used herein in reference toa clostridial toxin substrate, the term “in solution phase” means thatthe substrate is soluble and, during proteolysis, is not constrained orimmobilized on a solid support such as a column or dish.

In the methods of the invention, a sample is treated with a clostridialtoxin substrate under conditions suitable for clostridial toxin proteaseactivity. Exemplary conditions suitable for clostridial toxin proteaseactivity are well known in the art, and further can be determined byroutine methods. See, for example, Hallis et al., J. Clin. Microbiol.34:1934-1938 (1996); Ekong et al., Microbiol. 143:3337-3347 (1997);Shone et al., WO 95/33850; Schmidt and Bostian, supra, 1995; Schmidt andBostian, supra, 1997; Schmidt et al., supra, 1998; and Schmidt andBostian, U.S. Pat. No. 5,965,699. It is understood that conditionssuitable for clostridial toxin protease activity can depend, in part, onthe specific clostridial toxin type or subtype being assayed and thepurity of the toxin preparation. Conditions suitable for clostridialtoxin protease activity generally include a buffer, such as HEPES, Trisor sodium phosphate, typically in the range of pH 5.5 to 9.5, forexample, in the range of pH 6.0 to 9.0, pH 6.5 to 8.5 or pH 7.0 to 8.0.Conditions suitable for clostridial toxin protease activity also caninclude, if desired, dithiothreitol, β-mercaptoethanol or anotherreducing agent, for example, where a dichain toxin is being assayed(Ekong et al., supra, 1997). In one embodiment, the conditions includeDTT in the range of 0.01 mM to 50 mM; in other embodiments, theconditions include DTT in the range of 0.1 mM to 20 mM, 1 to 20 mM, or 5to 10 mM. If desired, an isolated clostridial toxin or sample can bepre-incubated with a reducing agent, for example, with 10 mMdithiothreitol (DTT) for about 30 minutes prior to addition ofclostridial toxin substrate.

Clostridial toxins are zinc metalloproteases, and a source of zinc, suchas zinc chloride or zinc acetate, typically in the range of 1 to 500 μM,for example, 5 to 10 μM can be included, if desired, as part of theconditions suitable for clostridial toxin protease activity. One skilledin the art understands that zinc chelators such as EDTA generally areexcluded from a buffer for determining the activity of a clostridialtoxin.

Conditions suitable for clostridial toxin protease activity canoptionally include a detergent such as TWEEN-20, which can be used, forexample, in place of bovine serum albumin. TWEEN-20 can be provided, forexample, in the range of 0.001% to 10% (v/v), or in the range of 0.01%to 1.0% (v/v). As a non-limiting example, TWEEN-20 can be included at aconcentration of 0.1% (v/v).

Conditions suitable for clostridial toxin protease activity also caninclude, if desired, bovine serum albumin (BSA) or another agent whichacts as a protein stabilizer, solubilizing agent or blocker of surfaceloss. As an example, when included, BSA typically is provided in therange of 0.1 mg/ml to 10 mg/ml. In one embodiment, BSA is included at aconcentration of 1 mg/ml. See, for example, Schmidt and Bostian, supra,1997. In another embodiment, BSA is included at a concentration of 0.1%(w/v).

The amount of clostridial toxin substrate can be varied in a method ofthe invention. A clostridial toxin substrate can be supplied, forexample, at a concentration of 1 μM to 500 μM, 1 μM to 50 μM, 1 μM to 30μM, 5 μM to 20 μM, 50 μM to 3.0 mM, 0.5 mM to 3.0 mM, 0.5 mM to 2.0 mM,or 0.5 mM to 1.0 mM. The skilled artisan understands that theconcentration of clostridial toxin substrate or the amount of sample canbe limited, if desired, such that the assay is linear. In oneembodiment, a method of the invention relies on a clostridial toxinsubstrate concentration of less than 100 μM. In further embodiments, amethod of the invention relies on a clostridial toxin substrateconcentration of less than 50 μM or less than 25 μM. In a furtherembodiment, a method of the invention relies on a clostridial toxinsubstrate concentration of 10 μM to 20 μM. If desired, a linear assayalso can be performed by mixing clostridial toxin substrate withcorresponding, “unlabeled” substrate which lacks a fluorophore. Theappropriate dilution can be determined, for example, by preparing serialdilutions of clostridial toxin substrate in the corresponding unlabeledsubstrate.

The concentration of purified or partially purified clostridial toxin tobe assayed in a method of the invention generally is in the range ofabout 0.0001 ng/ml to 500 μg/ml toxin, for example, about 0.0001 ng/mlto 50 μg/ml toxin, 0.001 ng/ml to 500 μg/ml toxin, 0.001 ng/ml to 50μg/ml toxin, 0.0001 to 5000 ng/ml toxin, for example, about 0.001 ng/mlto 5000 ng/ml, 0.01 ng/ml to 5000 ng/ml, 0.1 ng/ml to 5000 ng/ml, 1ng/ml to 5000 ng/ml, 10 ng/ml to 5000 ng/ml, 50 ng/ml to 5000 ng/ml, 50ng/ml to 500 ng/ml or 100 ng/ml to 5000 ng/ml toxin, which can be, forexample, purified recombinant dichain toxin or formulated clostridialtoxin product containing human serum albumin and excipients. Generally,the amount of purified toxin assayed in a method of the invention is inthe range of 0.1 pg to 100 μg, for example, 0.1 pg to 50 μg or 0.1 pg to10 μg.

The concentration of purified or partially purified clostridial toxinassayed in a method of the invention can be, for example, in the rangeof about 0.1 pM to 100 μM, 0.1 pM to 10 μM, 0.1 pM to 1 μM, 0.1 pM to500 nM, 0.1 pM to 100 nM, for example, 1 pM to 2000 pM, 1 pM to 200 pM,1 pM to 50 pM, 1 nM to 1 μM, 1 nM to 500 nM, 1 nM to 200 nM, 1 nM to 100nM or 3 nM to 100 nM toxin, which can be, for example, purified nativeor recombinant light chain or dichain toxin or formulated clostridialtoxin product containing human serum albumin and excipients. Inparticular embodiments, the concentration of purified or partiallypurified recombinant BoNT/A or BoNT/E light chain or dichain orformulated toxin product is in the range of 1 pM to 2000 pM, 10 pM to2000 pM, 20 pM to 2000 pM, 40 pM to 2000 pM, or 1 pM to 200 pM. Infurther embodiments, the concentration of purified or partially purifiedrecombinant BoNT/C light chain or dichain or formulated toxin product isin the range of 1 to 200 nM, 4 to 100 nM, 10 to 100 nM or 4 to 60 nM.One skilled in the art understands that the concentration of purified orpartially purified clostridial toxin will depend on the serotype of thetoxin assayed, as well as the purity or recombinant sequence of thetoxin, the presence of inhibitory components, and the assay conditions.It is additionally understood that purified, partially purified or crudesamples can be diluted to within a convenient range for assaying forclostridial toxin protease activity against a standard curve. Similarly,it is understood that a sample can be diluted, if desired, such that theassay is linear.

Conditions suitable for clostridial toxin protease activity alsogenerally include, for example, temperatures in the range of about 20°C. to about 45° C., for example, in the range of 25° C. to 40° C., orthe range of 35° C. to 39° C. Assay volumes often are in the range ofabout 5 to about 200 μl, for example, in the range of about 10 μl to 100μl or about 0.5 μl to 100 μl, although nanoliter reaction volumes alsocan be used with the methods of the invention. Assay volumes also canbe, for example, in the range of 100 μl to 2.0 ml or in the range of 0.5ml to 1.0 ml.

One skilled in the art understands that fluorescence polarizationreactions may or may not be terminated and that assay times can bevaried as appropriate by the skilled artisan. Assay times generallydepend, in part, on the concentration, purity and activity of theclostridial toxin and generally vary, without limitation, in the rangeof about 15 minutes to about 5 hours. As non-limiting examples,exemplary assay times include incubation, for example, at 37° C. for 30minutes, 45 minutes, 60 minutes, 75 minutes or 90 minutes. In particularembodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%or 100% of the clostridial toxin substrate is cleaved. In furtherembodiments, the protease reaction is stopped before more than 5%, 10%,15%, 20%, 25% or 50% of the clostridial toxin substrate is cleaved. Itis understood that protease reactions can be terminated by theappropriate reagent, which generally depends on the fluorophore andother components of the substrate. As a non-limiting example, a proteasereaction based on a substrate containing GFP as the donor fluorophorecan be terminated by the addition of guanidinium chloride, for example,to a final concentration of 1 to 2 M. Protease reactions also can beterminated by addition of H₂SO₄; addition of about 0.5 to 1.0 sodiumborate, pH 9.0 to 9.5; or addition of zinc chelators. One skilled in theart understands that protease reactions can be terminated, if desired,prior to exciting the fluorophore or donor fluorophore with planepolarized light.

As a non-limiting example, conditions suitable for clostridial toxinprotease activity such as BoNT/A protease activity can be incubation at37° C. for 90 minutes in a buffer containing 50 mM HEPES (pH 7.2), 10 μMZnCl₂, 10 mM DTT, and 0.1% (v/v) TWEEN-20 with 10-16 μM substrate. Ifdesired, samples containing BoNT/A, particularly dichain BoNT/A, can bepreincubated with dithiothreitol, for example, for 20 or 30 minutesbefore addition of substrate. As a further non-limiting example,conditions suitable for BoNT/A protease activity can be incubation at37° C. in a buffer such as 30 mM HEPES (pH 7.3) containing a reducingagent such as 5 mM dithiothreitol; and a source of zinc such as 25 μMzinc chloride (approximately 7 nM; Schmidt and Bostian, supra, 1997).BSA in the range of 0.1 mg/ml to 10 mg/ml, for example, 1 mg/ml BSA,also can be included when a sample is treated with a clostridial toxinsubstrate (Schmidt and Bostian, supra, 1997). As another non-limitingexample, conditions suitable for clostridial toxin protease activity,for example BoNT/B activity, can be incubation in 50 mM HEPES, pH 7.4,with 10 μM zinc chloride, 1% fetal bovine serum and 10 mMdithiothreitol, with incubation for 90 minutes at 37° C. (Shone andRoberts, Eur. J. Biochem. 225:263-270 (1994); Hallis et al., supra,1996); or can be, for example, incubation in 40 mM sodium phosphate, pH7.4, with 10 mM dithiothreitol, optionally including 0.2% (v/v) TritonX-100, with incubation for 2 hours at 37° C. (Shone et al., supra,1993). Conditions suitable for tetanus toxin protease activity or otherclostridial toxin protease activity can be, for example, incubation in20 mM HEPES, pH 7.2, and 100 mM NaCl for 2 hours at 37° C. with 25 μMpeptide substrate (Cornille et al., supra, 1994).

In one embodiment, conditions suitable for clostridial toxin proteaseactivity include cationic polyamino acids such as polyarginine in abuffer of suitable ionic strength. Where there is a charge difference inthe clostridial toxin substrate as compared to the cleavage product,fluorescence polarization can be observed in the presence ofpolyarginine or another cationic polyamino acid (Simeonov et al.,Analytical Biochemistry 304:193-199 (2002)). As a non-limiting example,if the net ionic charge of a fluorescently labeled cleavage productbecomes negative following treatment of clostridial toxin substrate witha toxin sample, polyarginine will selectively bind to the fluorescentlylabeled cleavage product, thereby generating a measurable increase inpolarization.

It is understood that any of a variety of control substrates are usefulin the methods of the invention. A control substrate can be, forexample, a clostridial toxin substrate which is not treated with active,toxin-containing sample; a polarization value determined before additionof the sample; or a similar, but different substrate which does notcontain a toxin cleavage site or functional recognition sequence.

It is understood that the methods of the invention can be automated andcan be configured in a high-throughput or ultra high-throughput formatusing, without limitation, 96-well, 384-well or 1536-well plates. Any ofa variety of spectrofluorometers equipped with an appropriate polarizercan be used to assay the change in fluorescence polarization over timeincluding, without limitation a Cary Eclipse spectrofluorometer; theBeckmann Affinity™ Multi-Mode plate reader; TECAN GeniusPro; and othersystems from, for example, Perkin Elmer.

Further provided herein are methods of determining the presence oractivity of a clostridial toxin by (a) treating with a sample, underconditions suitable for clostridial toxin protease activity, aclostridial toxin substrate containing (i) a donor fluorophore; (ii) anacceptor having an absorbance spectrum overlapping the emission spectrumof the donor fluorophore; and (iii) a clostridial toxin recognitionsequence containing a cleavage site, where the cleavage site intervenesbetween the donor fluorophore and the acceptor and where, under theappropriate conditions, resonance energy transfer is exhibited betweenthe donor fluorophore and the acceptor; (b) exciting the donorfluorophore with plane polarized light; and (c) determining fluorescencepolarization of the treated substrate relative to a control substrate,where a change in fluorescence polarization of the treated substrate ascompared to fluorescence polarization of the control substrate isindicative of the presence or activity of the clostridial toxin. In oneembodiment, step (c) includes determining the change in fluorescencepolarization of the treated substrate over time.

In the methods of the invention based on FRET-assisted fluorescencepolarization, the change in fluorescence polarization can be an increaseor decrease in fluorescence polarization. In one embodiment, the donorfluorophore has a fluorescence lifetime of at least 0.5 nanoseconds. Inanother embodiment, the donor fluorophore has a fluorescence lifetime ofat least 5 nanoseconds. A donor fluorophore useful in the invention canbe, without limitation, a green fluorescent protein (GFP); bluefluorescent protein (BFP); cyan fluorescent protein (CFP); yellowfluorescent protein (YFP); red fluorescent protein (RFP); Alexa Fluor®dye; fluorescein; a fluorescein derivative;diaminotriazinylamino-fluorescein (DTAF); a biarsenic derivative offluorescein; fluorescein arsenical hairpin binding dye (FlAsH™); redbiarsenical dye (ReAsH™); carboxyfluorescein (FAM); Texas Red™;tetramethylcarboxy-rhodamine (TMR); carboxy-x-rhodamine (ROX); rhodaminegreen; Oregon Green 488; BODIPY®-TR; BODIPY®-TMR; BODIPY®-FL; Cy3, Cy™3Bor Dansyl. In particular embodiments, the donor fluorophore is a greenfluorescent protein; blue fluorescent protein; cyan fluorescent protein;yellow fluorescent protein or red fluorescent protein. In oneembodiment, the donor fluorophore is a green fluorescent protein (GFP).In another embodiment, the acceptor fluorophore is Alexa Fluor® 546.

Any of a variety of recognition sequences can be included in aclostridial toxin substrate useful in a method of the invention. In oneembodiment, the recognition sequence is a BoNT/A recognition sequencesuch as, without limitation, a BoNT/A recognition sequence containing atleast six consecutive residues of SNAP-25, where the six consecutiveresidues include Gln-Arg, or a peptidomimetic thereof. Such a BoNT/Arecognition sequence can include, for example, residues 134 to 206 ofSEQ ID NO: 2. A recognition sequence included in a clostridial toxinsubstrate useful in a method of the invention also can be, withoutlimitation, a BoNT/B recognition sequence. Such a BoNT/B recognitionsequence can contain, for example, at least six consecutive residues ofVAMP, where the six consecutive residues include Gln-Phe, or apeptidomimetic thereof. In a further embodiment, a recognition sequenceincluded in a clostridial toxin substrate useful in a method of theinvention is a BoNT/C1 recognition sequence. Such a BoNT/C1 recognitionsequence can contain, without limitation, at least six consecutiveresidues of syntaxin, where the six consecutive residues includeLys-Ala, or a peptidomimetic thereof. A BoNT/C1 recognition sequenceuseful in the invention also can contain at least six consecutiveresidues of SNAP-25, where the six consecutive residues include Arg-Ala,or a peptidomimetic thereof.

In a further embodiment, a recognition sequence included in aclostridial toxin substrate useful in a method of the invention is aBoNT/D recognition sequence. Such a BoNT/D recognition sequence cancontain, for example, at least six consecutive residues of VAMP, wherethe six consecutive residues include Lys-Leu, or a peptidomimeticthereof. A recognition sequence useful in the invention also can be, forexample, a BoNT/E recognition sequence. Such a BoNT/E recognitionsequence can contain, without limitation, at least six consecutiveresidues of SNAP-25, where the six consecutive residues include Arg-Ile,or a peptidomimetic thereof. In yet another embodiment, a recognitionsequence included in a clostridial toxin substrate useful in a method ofthe invention is a BoNT/F recognition sequence. BoNT/F recognitionsequences useful in the invention encompass, without limitation, thosehaving at least six consecutive residues of VAMP, where the sixconsecutive residues include Gln-Lys, or a peptidomimetic thereof. Arecognition sequence included in a clostridial toxin substrate useful ina method of the invention also can be a BoNT/G recognition sequence.Such BoNT/G recognition sequences encompass, without limitation, thosehaving at least six consecutive residues of VAMP, where the sixconsecutive residues include Ala-Ala, or a peptidomimetic thereof. Instill a further embodiment, a recognition sequence included in aclostridial toxin substrate useful in a method of the invention is aTeNT recognition sequence. Such a TeNT recognition sequence can be,without limitation, a sequence containing at least six consecutiveresidues of VAMP, where the six consecutive residues include Gln-Phe, ora peptidomimetic thereof.

Any of a variety of clostridial toxin substrates can be useful in themethods of the invention, including peptides and peptidomimetics havingat least 100 residues, or having at least 200 residues. Furthermore, anyof a variety of samples can be assayed according to a method of theinvention including, without limitation, crude cell lysates, isolatedclostridial toxins including isolated clostridial toxin light chains;and formulated clostridial toxin products such as formulated BoNT/A,BoNT/B or BoNT/E toxin products.

Where a method of the invention involves fluorescence resonance energytransfer, the method relies on a clostridial toxin substrate whichincludes, in part, a donor fluorophore. Like a “fluorophore,” a “donorfluorophore” is a molecule that, when irradiated with light of a certainwavelength, emits light, also denoted fluorescence, of a differentwavelength. A donor fluorophore is a fluorophore which, when paired witha suitable acceptor, transfers energy to the acceptor.

As used herein, the term “acceptor” means a molecule that can absorbenergy from, and upon excitation of, a donor fluorophore. An acceptoruseful in a clostridial toxin substrate has an absorbance spectrum whichoverlaps the emission spectrum of a donor fluorophore included in thesubstrate. An acceptor useful in the invention generally has rather lowabsorption at a wavelength suitable for excitation of the donorfluorophore.

As set forth above, an acceptor has an absorbance spectrum that overlapsthe emission spectrum of the donor fluorophore. The term “overlapping,”as used herein in reference to the absorbance spectrum of an acceptorand the emission spectrum of a donor fluorophore, means an absorbancespectrum and emission spectrum that are partly or entirely shared. Thus,in such overlapping spectra, the high end of the range of the donorfluorophore's emission spectrum is higher than the low end of the rangeof the acceptor's absorbance spectrum.

As set forth above, any of a variety of donor fluorophores can be usefulin the invention, including, without limitation, green fluorescentprotein; blue fluorescent protein; cyan fluorescent protein; yellowfluorescent protein; red fluorescent protein; an Alexa Fluor® dye;fluorescein; a fluorescein derivative; diaminotriazinylamino-fluorescein(DTAF); a biarsenic derivative of fluorescein; fluorescein arsenicalhairpin binding dye (FlAsH™); red biarsenical dye (ReAsH™);carboxyfluorescein (FAM); Texas Red™; tetramethylcarboxy-rhodamine(TMR); carboxy-x-rhodamine (ROX); rhodamine green; Oregon Green 488;BODIPY®-TR; BODIPY®-TMR; BODIPY®-FL; Cy3, Cy™3B or Dansyl. A variety ofacceptors also can be useful in the invention including, but not limitedto, Alexa Fluor® dyes such as Alexa Fluor® 546, Alexa Fluor® 568, AlexaFluor® 610, Alexa Fluor® 660 and Alexa Fluor® 750; QSY® 7;tetramethylrhodamine; octadecylrhodamine; flavodoxin, cytochrome cperoxidase; and rubredoxin.

Exemplary donor fluorophore-acceptor pairs which exhibit FRET and areuseful in the methods of the invention encompass, without limitation,GFP and Alexa Fluor® 546; fluorescein and QSY® 7; fluorescein andtetramethylrhodamine; and dansyl and octadecylrhodamine. Furtherexemplary donor fluorophore-acceptor pairs which are useful in themethods of the invention encompass, without limitation, Alexa Fluor® 633and Alexa Fluor® 660; Alexa Fluor® 594 and Alexa Fluor® 610; AlexaFluor® 700 and Alexa Fluor® 750; and Alexa Fluor® 555 and Alexa Fluor®568. Additional acceptors useful in the invention include those in whichthe acceptor is a protein with a visible chromophore such as, withoutlimitation, flavodoxin, cytochrome c peroxidase or rubredoxin; such aprotein can have, for example, a molecular weight in the range of 6 to34 kDa and a chromophore which absorbs strongly in the region between400-500 nm. Exemplary donor fluorophore-acceptor pairs based on suchproteins include, but are not limited to,5-(((2-iodoaacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5IAEDANS) and flavodoxin; 4-acetamido-4′ maleimidylstilbene 2,2′disulfonic acid and cytochrom c peroxidase; and Alexa Fluor®488 andrubredoxin. These and other donor fluorophores suitable for fluorescencepolarization can be paired with any of a variety of acceptors having anabsorbance spectrum which overlaps the emission spectrum of the donorfluorophore.

One skilled in the art understands that the methods of the inventionbased on FRET-assisted fluorescence polarization optionally utilize asubstrate which includes a bulking group in addition to the donorfluorophore and acceptor. One skilled in the art understands that theoptional inclusion of a bulking group depends on the molecular weightand bulking characteristics of the selected donor fluorophore andacceptor. A variety of bulking groups are optionally useful in theinvention, including those described herein above.

Substrates useful in the invention can be prepared by recombinantmethods or using synthetic chemical methods, or a combination thereof.As described herein in Example I, a fusion protein containing a bulkinggroup fused to a BoNT/A clostridial toxin recognition sequence and acarboxy-terminal cysteine was prepared by recombinant methods. Thecarboxy-terminal cysteine was used for attachment of a fluorophore toproduce the complete clostridial toxin substrate. Recombinant methodsfor preparation of clostridial toxin substrates which are fusionproteins are well known in the art as described, for example, inAusubel, Current Protocols in Molecular Biology John Wiley & Sons, Inc.,New York 2000.

Chemical methods for modifying a protein, peptide or peptidomimetic tocontain a fluorophore and bulking group, or a donor fluorophore andacceptor, are well known in the art (Fairclough and Cantor, MethodsEnzymol. 48:347-379 (1978); Glaser et al., Chemical Modification ofProteins Elsevier Biochemical Press, Amsterdam (1975); Haugland, ExcitedStates of Biopolymers (Steiner Ed.) pp. 29-58, Plenum Press, New York(1983); Means and Feeney, Bioconjugate Chem. 1:2-12 (1990); Matthews etal., Methods Enzymol. 208:468-496 (1991); Lundblad, Chemical Reagentsfor Protein Modification 2nd Ed., CRC Press, Boca Ratan, Fla. (1991);Haugland, supra, 1996). A variety of groups can be used to couple afluorophore, bulking group, donor fluorophore or acceptor, for example,to a peptide or peptidomimetic containing a clostridial toxinrecognition sequence. A thiol group, for example, can be used to couplea fluorophore, bulking group, donor fluorophore or acceptor to thedesired position in a peptide or peptidomimetic to produce a clostridialtoxin substrate useful in the invention (see Example I). Haloacetyl andmaleimide labeling reagents also can be used to couple a fluorophore,bulking group, donor fluorophore or acceptor in preparing a clostridialtoxin substrate useful in the invention. See, for example, Wu and Brand,supra, 1994.

Cross-linker moieties also can be useful for preparing a clostridialtoxin substrate. Cross-linkers are well known in the art and includehomo- and hetero-bifunctional cross-linkers such as BMH and SPDP. Wherea fluorophore, bulking group, donor fluorophore or acceptor is aprotein, well known chemical methods for specifically linking moleculesto the amino- or carboxy-terminus of a protein can be employed. See, forexample, “Chemical Approaches to Protein Engineering” in ProteinEngineering: A Practical Approach Rees et al. (Eds) Oxford UniversityPress, 1992.

Where a clostridial toxin substrate contains a fluorophore and bulkinggroup, the clostridial toxin cleavage site is positioned between thefluorophore and bulking group. In one embodiment, the fluorophore ispositioned carboxy-terminal of the cleavage site while the bulking groupis positioned amino-terminal of the cleavage site. In anotherembodiment, the fluorophore is positioned amino-terminal of the cleavagesite while the bulking group is positioned carboxy-terminal of thecleavage site.

Where a clostridial toxin substrate contains a donor fluorophore and anacceptor, the clostridial toxin cleavage site is positioned between thedonor fluorophore and the acceptor. In one embodiment, the donorfluorophore is positioned amino-terminal of the cleavage site while theacceptor is positioned carboxy-terminal of the cleavage site. In anotherembodiment, the donor fluorophore is positioned carboxy-terminal of thecleavage site while the acceptor is positioned amino-terminal of thecleavage site.

One skilled in the art understands that there are several considerationsin selecting and positioning a fluorophore and a bulking group, or adonor fluorophore and an acceptor, in a clostridial toxin substrateuseful in the invention. The fluorophore and bulking group, or donorfluorophore and acceptor, generally are positioned to minimizeinterference with substrate binding to, or proteolysis by, theclostridial toxin. Thus, a fluorophore and bulking group, or donorfluorophore and acceptor, can be selected and positioned, for example,so as to minimize the disruption of bonded and non-bonded interactionsthat are important for binding, and to minimize steric hindrance. Inaddition, as discussed further below, the spatial distance between anacceptor and donor fluorophore generally is limited to achieve efficientenergy transfer from the donor fluorophore to the acceptor.

As discussed above, efficiency of energy transfer from a donorfluorophore to an acceptor is dependent, in part, on the spatialseparation of the donor fluorophore and acceptor molecules. As thedistance between the donor fluorophore and acceptor increases, there isless energy transfer to the acceptor, and the donor fluorescence signaltherefore increases. The overall energy transfer between the donorfluorophore and acceptor is dependent upon many factors, including theseparation distance between the donor fluorophore and acceptor in thesubstrate, the spectral overlap between donor fluorophore and acceptor,and the substrate concentration. One skilled in the art understandsthat, as the concentration of substrate increases, intermolecularquenching of the donor, even after proteolytic cleavage, can become afactor. This phenomenon is denoted the “inner filter effect.” Oneskilled in the art further understands that the concentration ofsubstrate can be controlled as described above.

The Förster distance, which is the separation between a donorfluorophore and an acceptor for 50% energy transfer, represents aspatial separation between donor fluorophore and acceptor that providesa good sensitivity. For peptide substrates, adjacent residues areseparated by a distance of approximately 3.6 Å in the most extendedconformation. For example, the calculated Förster distance for afluorescein/tetramethylrhodamine pair is 55 Å, which would represent aspatial separation between fluorescein and tetramethylrhodamine of about15 residues in the most extended conformation. Because peptides andpeptidomimetics in solution rarely have a fully extended conformation,donor fluorophores and acceptors can be more widely separated thanexpected based on a calculation performed using 3.6 Å per residue andstill remain within the Förster distance as shown, for example, by theoccurrence of FRET between donor-acceptor pairs separated by about 50amino acids (Graham et al., Analyt. Biochem. 296: 208-217 (2001)).

Förster theory is based on very weak interactions between a donorfluorophore and an acceptor; spectroscopic properties such as absorptionof one fluorophore should not be altered in the presence of the other,defining the shortest distance range over which the theory is valid. Itis understood that, for many donor fluorophore-acceptor pairs, Förstertheory is valid when donor fluorophores and acceptors are separated byabout 10 Å to 100 Å. However, for particular donor fluorophore-acceptorpairs, Förster theory is valid below 10 Å as determined by subpicosecondtechniques (Kaschke and Ernsting, Ultrafast Phenomenon in Spectroscopy(Klose and Wilhelmi (Eds.)) Springer-Verlag, Berlin 1990).

In particular embodiments, the invention provides a method that relieson a clostridial toxin substrate in which the donor fluorophore isspatially separated from the acceptor by a distance of at most 100 Å. Inother embodiments, the invention provides a method that relies on aclostridial toxin substrate in which the donor fluorophore is spatiallyseparated from the acceptor by a distance of at most 90 Å, 80 Å, 70 Å,60 Å, 50 Å, 40 Å, 30 Å or 20 Å. In further embodiments, the inventionprovides a method that relies on a clostridial toxin substrate in whichthe donor fluorophore is spatially separated from the acceptor by adistance of 10 Å to 100 Å, 10 Å to 80 Å, 10 Å to 60 Å, 1A to 40 Å, 10 Åto 20 Å, 20 Å to 100 Å, 20 Å to 80 Å, 20 Å to 60 Å, 20 Å to 40 Å, 40 Åto 100 Å, 40 Å to 80 Å or 40 Å to 60 Å. In still further embodiments,the invention provides a method that relies on a clostridial toxinsubstrate in which the donor fluorophore and the acceptor are separatedin the primary amino acid sequence by at most six residues, at mosteight residues, at most ten residues, at most twelve residues, at mostfifteen residues, at most twenty residues, at most twenty-five residues,at most thirty residues, at most thirty-five residues, at most fortyresidues, at most forty-five residues, at most fifty residues, at mostsixty residues, at most seventy residues, at most eighty residues, atmost ninety residues, at most 100 residues, at most 150 residues, atmost 200 residues or up to the full-length of a naturally occurringclostridial toxin target protein.

One skilled in the art understands that a clostridial toxin substrateuseful in the invention can be designed, if desired, to optimize theefficiency of FRET. One skilled in the art understands that a donorfluorophore can be selected, if desired, with a high quantum yield, andacceptor can be selected, if desired, with a high extinction coefficientto maximize the Förster distance. One skilled in the art furtherunderstands that fluorescence arising from direct excitation of anacceptor can be difficult to distinguish from fluorescence resultingfrom resonance energy transfer. Thus, it is recognized that a donorfluorophore and acceptor can be selected which have relatively littleoverlap of their excitation spectra such that the donor can be excitedat a wavelength that does not result in direct excitation of theacceptor. It further is recognized that a clostridial toxin substrateuseful in the invention can be designed so that the emission spectra ofthe donor fluorophore and acceptor overlap relatively little such thatthe two emissions can be readily distinguished.

Specific and distinct cleavage sites for different clostridial toxinsare well known in the art. BoNT/A cleaves a Gln-Arg bond; BoNT/B andTeNT cleave a Gln-Phe bond; BoNT/C1 cleaves a Lys-Ala or Arg-Ala bond;BoNT/D cleaves a Lys-Leu bond; BoNT/E cleaves an Arg-Ile bond; BoNT/Fcleaves a Gln-Lys bond; and BoNT/G cleaves an Ala-Ala bond (see TableA). In standard nomenclature, the sequence surrounding a clostridialtoxin cleavage site is denoted P₅-P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′-P₅′ withP₁-P₁′ representing the scissile bond. It is understood that a P₁ or P₁′site, or both, can be substituted with another amino acid or amino acidmimetic in place of the naturally occurring residue. As an example,BoNT/A substrates have been prepared in which the P₁ position (Gln) ismodified to be an alanine, 2-aminobutyric acid or asparagine residue;these substrates were hydrolyzed by BoNT/A at the P₁Arg bond (Schmidtand Bostian, J. Protein Chem. 16:19-26 (1997)). While it is recognizedthat substitutions can be introduced at the P₁ position of the scissilebond, for example, a BoNT/A scissile bond, it is further recognized thatconservation of the P₁′ residue can be advantageous (Vaidyanathan etal., J. Neurochem. 72:327-337 (1999)). Thus, in particular embodiments,the invention provides a method which relies on a clostridial toxinsubstrate having a clostridial toxin recognition sequence in which theP₁′ residue is not modified or substituted relative to the naturallyoccurring residue in a target protein cleaved by the clostridial toxin.In other embodiments, the invention provides a method which relies on aclostridial toxin substrate having a recognition sequence in which theP₁ residue is modified or substituted relative to the naturallyoccurring residue in a target protein cleaved by the clostridial toxin;such a clostridial toxin substrate retains susceptibility to peptidebond cleavage between the P₁ and P₁′ residues.

TABLE A BONDS CLEAVED IN HUMAN VAMP-2, SNAP-25 OR SYNTAXIN SEQ ID ToxinTarget P₄-P₃-P₂-P₁-P₁′-P₂′-P₃′-P₄′ NO: BoNT/ SNAP-25Glu-Ala-Asn-Gln-Arg*-Ala-Thr-Lys 22 A BoNT/ VAMP-2Gly-Ala-Ser-Gln-Phe*-Glu-Thr-Ser 23 B BoNT/ syntaxinAsp-Thr-Lys-Lys-Ala*-Val-Lys-Tyr 24 C1 BoNT/ VAMP-2Arg-Asp-Gln-Lys-Leu*-Ser-Glu-Leu 25 D BoNT/ SNAP-25Gln-Ile-Asp-Arg-Ile*-Met-Glu-Lys 26 E BoNT/ VAMP-2Glu-Arg-Asp-Gln-Lys*-Leu-Ser-Glu 27 F BoNT/ VAMP-2Glu-Thr-Ser-Ala-Ala*-Lys-Leu-Lys 28 G TeNT VAMP-2Gly-Ala-Ser-Gln-Phe*-Glu-Thr-Ser 29 *Scissile bond shown in bold

SNAP-25, VAMP and syntaxin share a short motif located within regionspredicted to adopt an α-helical conformation. This motif is present inSNAP-25, VAMP and syntaxin isoforms expressed in animals sensitive tothe neurotoxins. In contrast, Drosophila and yeast homologs that areresistant to these neurotoxins and syntaxin isoforms not involved inexocytosis contain sequence variations in the α-helical motif regions ofthese VAMP and syntaxin proteins.

Multiple repetitions of the α-helical motif are present in proteinssensitive to cleavage by clostridial toxins: Four copies are naturallypresent in SNAP-25; two copies are naturally present in VAMP; and twocopies are naturally present in syntaxin. Furthermore, peptidescorresponding to the specific sequence of the α-helical motifs caninhibit neurotoxin activity in vitro and in vivo, and such peptides cancross-inhibit different neurotoxins. In addition, antibodies raisedagainst such peptides can cross-react among the three target proteins,indicating that this α-helical motif is exposed on the protein surfaceand adopts a similar configuration in each of the three target proteins.Consistent with these findings, SNAP-25-specific, VAMP-specific andsyntaxin-specific neurotoxins cross-inhibit each other by competing forthe same binding site, although they do not cleave targetsnon-specifically. These results indicate that a clostridial toxinrecognition sequence can include, if desired, at least one α-helicalmotif. It is recognized that an α-helical motif is not required forcleavage by a clostridial toxin, as evidenced by 16-mer and 17-mersubstrates for BoNT/A known in the art.

Although multiple α-helical motifs are found in the naturally occurringSNAP-25, VAMP and syntaxin target proteins, a clostridial toxinrecognition sequence useful in a clostridial toxin substrate can have asingle α-helical motif. In particular embodiments, a method of theinvention relies on a clostridial toxin recognition sequence includingtwo or more α-helical motifs. A BoNT/A or BoNT/E recognition sequencecan include, for example, the S4 α-helical motif, alone or combined withone or more additional α-helical motifs; a BoNT/B, BoNT/G or TeNTrecognition sequence can include, for example, the V2 α-helical motif,alone or combined with one or more additional α-helical motifs; aBoNT/C1 recognition sequence can include, for example, the S4 α-helicalmotif, alone or combined with one or more additional α-helical motifs,or the X2 α-helical motif, alone or combined with one or more additionalα-helical motifs; and a BoNT/D or BoNT/F recognition sequence caninclude, for example, the V1 α-helical motif, alone or combined with oneor more additional α-helical motifs.

As used herein, the term “botulinum toxin serotype A recognitionsequence” is synonymous with “BoNT/A recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements, or both, sufficient for detectable proteolysis at the scissilebond by a BoNT/A under conditions suitable for clostridial toxinprotease activity. A scissile bond cleaved by BoNT/A can be, forexample, Gln-Arg.

A variety of BoNT/A recognition sequences are well known in the art andare useful in the invention. A BoNT/A recognition sequence can have, forexample, residues 134 to 206 or residues 137 to 206 of human SNAP-25(Ekong et al., supra, 1997; U.S. Pat. No. 5,962,637). A BoNT/Arecognition sequence also can include, without limitation, the sequenceThr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQ ID NO: 30) or apeptidomimetic thereof, which corresponds to residues 190 to 202 ofhuman SNAP-25;Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys (SEQ ID NO:31) or a peptidomimetic thereof, which corresponds to residues 187 to201 of human SNAP-25;Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQ IDNO: 32) or a peptidomimetic thereof, which corresponds to residues 187to 202 of human SNAP-25;Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu (SEQID NO: 33) or a peptidomimetic thereof, which corresponds to residues187 to 203 of human SNAP-25;Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQID NO: 34) or a peptidomimetic thereof, which corresponds to residues186 to 202 of human SNAP-25; orAsp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu(SEQ ID NO: 35) or a peptidomimetic thereof, which corresponds toresidues 186 to 203 of human SNAP-25. See, for example, Schmidt andBostian, J. Protein Chem. 14:703-708 (1995); Schmidt and Bostian, supra,1997; Schmidt et al., FEBS Letters 435:61-64 (1998); and Schmidt andBostian, U.S. Pat. No. 5,965,699). If desired, a similar BoNT/Arecognition sequence can be prepared from a corresponding (homologous)segment of another BoNT/A-sensitive SNAP-25 isoform or homolog such as,for example, murine, rat, goldfish or zebrafish SNAP-25 or can be any ofthe peptides described herein or known in the art, for example, in U.S.Pat. No. 5,965,699.

A BoNT/A recognition sequence useful in the invention can correspond toa segment of a protein that is sensitive to cleavage by botulinum toxinserotype A, or can be substantially similar to a segment of aBoNT/A-sensitive protein. As illustrated in Table B, a variety ofnaturally occurring proteins sensitive to cleavage by BoNT/A are knownin the art and include, for example, human, mouse and rat SNAP-25; andgoldfish SNAP-25A and SNAP-25B. Thus, a BoNT/A recognition sequenceuseful in the invention can correspond, for example, to a segment ofhuman SNAP-25, mouse SNAP-25, rat SNAP-25, goldfish SNAP-25A or 25B, oranother naturally occurring protein sensitive to cleavage by BoNT/A.Furthermore, comparison of native SNAP-25 amino acid sequences cleavedby BoNT/A reveals that such sequences are not absolutely conserved (seeTable B and FIG. 3), indicating that a variety of amino acidsubstitutions and modifications relative to a naturally occurringBoNT/A-sensitive SNAP-25 sequence can be tolerated in a BoNT/Arecognition sequence useful in the invention.

TABLE B Cleavage of SNAP-25 and related proteins^(a,b,c,d) SEQ IDResistance to Species-Isoform Cleavage Sites NO: Cleavage by BoNT/EBoNT/A BoNT/C       

      

human 174                               206 mouse-SNAP-25 qnrqid rimekadsnktridean qra tkmlgsg none^(a) rat180                               end human-SNAP-23 qnpqik ritdkadtnrdridian ara kklids all^(b) 179                               endmouse-SNAP-23 qnqqiq ki tekadtnknridian tra kklids BoNT/A & C174                               end chicken-SNAP-25 qnrqid rimeklipikpglmkpt svq qrcsavvk BoNT/A & C171                               end goldfish-SNAP-25A qnrqid rimdmadsnktridean qra tkmlgsg none 172                               endgoldfish-SNAP-25B qnrqid ri mekadsnktridean qra tkmlgsg none180                               end Torpedo-SNAP-25 qnaqvd rivvkgdmnkaridean kha tkml BoNT/E^(c) & A180                               end sea urchin-SNAP-25 qnsqvg ritskaesnegrinsad kra knilrnk (?)^(e)203                               end C-elegans-SNAP-25 qnrqld rihdkqsnevrvesank rak nlitk BoNT/A & C182                               end Drosophila-SNAP-25 qnrqid rinrkgesneariavan qra hqllk BoNT/E & A^(e)181                               end leech-SNAP-25 qnrqvd rinnkmtsnqlrisdan kra skllke BoNT/A^(e) ^(a)= In vitro cleavage of SNAP-25requires 1000-fold higher BoNT/C concentration than BoNT/A or /E.^(b)= Substitution of p182r, or k185dd (boxes) induces susceptibilitytoward BoNT/E. ^(c)= Resistance to BoNT/A possibly due to d189 or e189substitution by v189, see box. ^(d)= Note that Torpedo is susceptible toBoNT/A. ^(e)= Note the presence of several non-conservative mutationsaround putative cleavage sites.

A clostridial toxin substrate, such as a substrate containing a BoNT/Arecognition sequence, can have one or multiple modifications as comparedto a naturally occurring sequence that is cleaved by the correspondingclostridial toxin. As an example, as compared to a 17-mer correspondingto residues 187 to 203 of human SNAP-25, substitution of Asp193 with Asnin the BoNT/A substrate resulted in a relative rate of proteolysis of0.23; substitution of Glu194 with Gln resulted in a relative rate of2.08; substitution of Ala195 with 2-aminobutyric acid resulted in arelative rate of 0.38; and substitution of Gln197 with Asn,2-aminobutyric acid or Ala resulted in a relative rate of 0.66, 0.25, or0.19, respectively (see Table C). Furthermore, substitution of Ala199with 2-aminobutyric acid resulted in a relative rate of 0.79;substitution of Thr200 with Ser or 2-aminobutyric acid resulted in arelative rate of 0.26 or 1.20, respectively; substitution of Lys201 withAla resulted in a relative rate of 0.12; and substitution of Met202 withAla or norleucine resulted in a relative rate of 0.38 or 1.20,respectively. See Schmidt and Bostian, supra, 1997. These resultsindicate that a variety of residues can be substituted in a clostridialtoxin substrate as compared to a naturally occurring toxin-sensitivesequence. In the case of BoNT/A, these results indicate that residuesincluding but not limited to Glu194, Ala195, Gln197, Ala199, Thr200 andMet202, Leu203, Gly204, Ser205, and Gly206, as well as residues moredistal from the Gln-Arg scissile bond, can be substituted or conjugatedto a fluorophore, bulking group, donor fluorophore or acceptor in aBoNT/A substrate useful in the invention. Such a BoNT/A substrate isdetectably proteolyzed at the scissile bond by BoNT/A under conditionssuitable for clostridial toxin protease activity. Thus, a BoNT/Asubstrate can include, if desired, one or several amino acidsubstitutions, additions or deletions relative to a naturally occurringSNAP-25 sequence.

TABLE C KINETIC PARAMETERS OF BONT/A SYNTHETIC PEPTIDE SUBSTRATESRelative Peptide Sequence^(a) SEQ ID NO: Rate^(b) [1-15] SNKTRIDEANQRATK31 0.03 [1-16] SNKTRIDEANQRATKM 32 1.17 [1-17] SNKTRIDEANQRATKML 33 1.00M16A SNKTRIDEANQRATK A L 50 0.38 M16X SNKTRIDEANQRATK X L 51 1.20 K15ASNKTRIDEANQRAT A ML 52 0.12 T14S SNKTRIDEANQRA S KML 53 0.26 T14BSNKTRIDEANQRA B KML 54 1.20 A13B SNKTRIDEANQR B TKML 55 0.79 Q11ASNKTRIDEAN A RATKML 56 0.19 Q11B SNKTRIDEAN B RATKML 57 0.25 Q11NSNKTRIDEAN N RATKML 58 0.66 N10A SNKTRIDEA A QRATKML 59 0.06 A9BSNKTRIDE B NQRATKML 60 0.38 E8Q SNKTRID Q ANQRATKML 61 2.08 D7N SNKTRI NEANQRATKML 62 0.23 ^(a)Nonstandard amino acid abbreviations are: B,2-aminobutyric acid; X, 2-aminohexanoic acid (norleucine) ^(b)Initialhydrolysis rates relative to peptide [1-17]. Peptide concentrations were1.0 mM.

As used herein, the term “botulinum toxin serotype B recognitionsequence” is synonymous with “BoNT/B recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements, or both, sufficient for detectable proteolysis at the scissilebond by a BoNT/B under appropriate conditions. A scissile bond cleavedby BoNT/B can be, for example, Gln-Phe.

A variety of BoNT/B recognition sequences are well known in the art orcan be defined by routine methods. Such BoNT/B recognition sequences caninclude, for example, a sequence corresponding to some or all of thehydrophilic core of a VAMP protein such as human VAMP-1 or human VAMP-2.A BoNT/B recognition sequence can include, without limitation, residues33 to 94, residues 45 to 94, residues 55 to 94, residues 60 to 94,residues 65 to 94, residues 60 to 88 or residues 65 to 88 of humanVAMP-2 (SEQ ID NO: 8), or residues 60 to 94 of human VAMP-1 (SEQ ID NO:7). See, for example, Shone et al., Eur. J. Biochem. 217: 965-971(1993). and U.S. Pat. No. 5,962,637. If desired, a similar BoNT/Brecognition sequence can be prepared from a corresponding (homologous)segment of another BoNT/B-sensitive VAMP isoform or homolog such ashuman VAMP-1 or rat or chicken VAMP-2.

Thus, it is understood that a BoNT/B recognition sequence can correspondto a segment of a protein that is sensitive to cleavage by botulinumtoxin serotype B, or can be substantially similar to such a segment of aBoNT/B-sensitive protein. As shown in Table D, a variety of naturallyoccurring proteins sensitive to cleavage by BoNT/B are known in the artand include, for example, human, mouse and bovine VAMP-1 and VAMP-2; ratVAMP-2; rat cellubrevin; chicken VAMP-2; Torpedo VAMP-1; sea urchinVAMP; Aplysia VAMP; squid VAMP; C. elegans VAMP; Drosophila n-syb; andleech VAMP. Thus, a BoNT/B recognition sequence included in a BoNT/Bsubstrate can correspond, for example, to a segment of human VAMP-1 orVAMP-2, mouse VAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2, rat VAMP-2, ratcellubrevin, chicken VAMP-2, Torpedo VAMP-1, sea urchin VAMP, AplysiaVAMP, squid VAMP, C. elegans VAMP, Drosophila n-syb, leech VAMP, oranother naturally occurring protein sensitive to cleavage by BoNT/B.Furthermore, as shown in Table D, comparison of native VAMP amino acidsequences cleaved by BoNT/B reveals that such sequences are notabsolutely conserved (see, also, FIG. 4), indicating that a variety ofamino acid substitutions and modifications relative to a naturallyoccurring VAMP sequence can be tolerated in a BoNT/B substrate of theinvention.

TABLE D Cleavage of VAMP^(a,b) SEQ ID Resistance to Species-IsoformCleavage Sites NO: Cleavage by               BoNT/B BoNT/F BoNT/D TeNTBoNT/G       

      

    

human 53                                          92 none mouse-VAMP-1dkvlerd qkl selddradalqagas qf ess aa klkrkyww bovine human51                                          90 none mouse-VAMP-2 dkvlerdqkl selddradalqagas qf ets aa klkrkyww bovine53                                          92 TeNT & rat-VAMP-2 dkvlerdqkl selddradalqagas vf ess aa klkrkyww BoNT/B51                                          90 rat-VAMP-2 dkvlerd qklselddradalqagas qf ets aa klkrkyww none38                                          77 rat-Cellubrevin dkvlerdqkl selddradalqagas qf ets aa klkrkyww none146                                        175 rat-TI-VAMP dlvaqrg erlellidktenlvdssv tf ktt sr nlaramcm all—                                            — TeNT & chicken-VAMP-1----erd qkl selddradalqagas vf ess aa klkr---- BoNT/B—                                            — chicken-VAMP-2 ----erdqkl selddradalqagas qf ets aa klkr---- none55                                          94 Torpedo-VAMP-1 dkvlerdqkl selddradalqagas qf ess aa klkrkyww none35                                          74 BoNT/F, D sea urchin-VAMPdkvldrd qal svlddradalqqgas qf etn ag klkrkyww & G41                                          80 Aplysia-VAMP ekvldrd qkisqlddraealqagas qf eas ag klkrkyww BoNT/G60                                          99 BoNT F & squid-VAMPdkvlerd ski selddradalqagas qf eas ag klkrkfww G86                                         115 BoNT/F, D C. elegans-VAMPnkvmerd vql nsldhraevlqngas qf qqs sr elkrqyww & G67                                         106 TeNT & Drosphila-syb^(a)ekvlerd qkl selgeradqleqgas qs eqq ag klkrkqww BoNT/B & G61                                         100 BoNT F &Drosphila-n-syb^(b) ekvlerd skl selddradalqqgas qf eqq ag klkrkfwl G49                                          88 leech-VAMP dkvlekd qklaeldgradalqagas qf eas ag klkrkfww BoNT/G ^(a)= Sequence corrected inposition 93 (f > s). ^(b)= Sequence corrected in position 68 (t > s).

As used herein, the term “botulinum toxin serotype C1 recognitionsequence” is synonymous with “BoNT/C1 recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements, or both, sufficient for detectable proteolysis at the scissilebond by a BoNT/C1 under appropriate conditions. A scissile bond cleavedby BoNT/C1 can be, for example, Lys-Ala or Arg-Ala.

It is understood that a BoNT/C1 recognition sequence can correspond to asegment of a protein that is sensitive to cleavage by botulinum toxinserotype C1, or can be substantially similar to a segment of aBoNT/C1-sensitive protein. As shown in Table E, a variety of naturallyoccurring proteins sensitive to cleavage by BoNT/C1 are known in the artand include, for example, human, rat, mouse and bovine syntaxin 1A and1B; rat syntaxins 2 and 3; sea urchin syntaxin; Aplysia syntaxin 1;squid syntaxin; Drosophila Dsynt1; and leech syntaxin 1. Thus, a BoNT/C1recognition sequence useful in a BoNT/C1 substrate can correspond, forexample, to a segment of human, rat, mouse or bovine syntaxin 1A or 1B,rat syntaxin 2, rat syntaxin 3, sea urchin syntaxin, Aplysia syntaxin 1,squid syntaxin, Drosophila Dsynt1, leech syntaxin 1, or anothernaturally occurring protein sensitive to cleavage by BoNT/C1.Furthermore, comparison of native syntaxin amino acid sequences cleavedby BoNT/C1 reveals that such sequences are not absolutely conserved (seeTable E and FIG. 5), indicating that a variety of amino acidsubstitutions and modifications relative to a naturally occurringBoNT/C1-sensitive syntaxin sequence can be tolerated in a BoNT/C1substrate useful in the invention.

TABLE E Cleavage of syntaxin Resis- tance SEQ to ID Cleav-Species-Isoform Cleavage Sites NO: age by BoNT/C

human 245              262 rat-syntaxin 1A eravsdtk ka vkyqskar no mousebovine human 244              261 rat-syntaxin 1B eravsdtk ka vkyqskarno mouse bovine 245              262 rat-syntaxin 2 ehakeetk ka ikyqskarno 244              261 rat-syntaxin 3 ekardetr ka mkyqgqar no244              261 rat-syntaxin 4 ergqehvk ia lenqkkar yes239              259 chicken-syntaxin 1B vpevfvtk sa vmyqcksr ex- pected243              260 sea urchin-syntaxin vrrqndtk ka vkyqskar no247              264 Aplysia-syntaxin 1 etakmdtk ka vkyqskar no248              265 squid-syntaxin etakvdtk ka vkyqskar no248              265 Drosophila-Dsynt 1 qtatqdtk ka lkyqskar no251              268 leech-syntaxin 1 etaaadtk ka mkyqsaar no

A variety of naturally occurring SNAP-25 proteins also are sensitive tocleavage by BoNT/C1, including human, mouse and rat SNAP-25; goldfishSNAP-25A and 25B; and Drosophila and leech SNAP-25. Thus, a BoNT/C1recognition sequence useful in a BoNT/C1 substrate can correspond, forexample, to a segment of human, mouse or rat SNAP-25, goldfish SNAP-25Aor 25B, Torpedo SNAP-25, zebrafish SNAP-25, Drosophila SNAP-25, leechSNAP-25, or another naturally occurring protein sensitive to cleavage byBoNT/C1. As discussed above in regard to variants of naturally occurringsyntaxin sequences, comparison of native SNAP-25 amino acid sequencescleaved by BoNT/C1 reveals significant sequence variability (see FIG. 3and Table B above), indicating that a variety of amino acidsubstitutions and modifications relative to a naturally occurringBoNT/C1-sensitive SNAP-25 sequence can be tolerated in a BoNT/C1substrate useful in the invention.

The term “botulinum toxin serotype D recognition sequence” is synonymouswith “BoNT/D recognition sequence” and means a scissile bond togetherwith adjacent or non-adjacent recognition elements, or both, sufficientfor detectable proteolysis at the scissile bond by a BoNT/D underappropriate conditions. A scissile bond cleaved by BoNT/D can be, forexample, Lys-Leu.

A variety of BoNT/D recognition sequences are well known in the art orcan be defined by routine methods. A BoNT/D recognition sequence caninclude, for example, residues 27 to 116; residues 37 to 116; residues 1to 86; residues 1 to 76; or residues 1 to 69 of rat VAMP-2 (SEQ ID NO:90; Yamasaki et al., J. Biol. Chem. 269:12764-12772 (1994)). Thus, aBoNT/D recognition sequence can include, for example, residues 27 to 69or residues 37 to 69 of rat VAMP-2 (SEQ ID NO: 90). If desired, asimilar BoNT/D recognition sequence can be prepared from a corresponding(homologous) segment of another BoNT/D-sensitive VAMP isoform or homologsuch as human VAMP-1 or human VAMP-2.

A BoNT/D recognition sequence can correspond to a segment of a proteinthat is sensitive to cleavage by botulinum toxin serotype D, or can besubstantially similar to a segment of a BoNT/D-sensitive protein. Asshown in Table D, a variety of naturally occurring proteins sensitive tocleavage by BoNT/D are known in the art and include, for example, human,mouse and bovine VAMP-1 and VAMP-2; rat VAMP-1 and VAMP-2; ratcellubrevin; chicken VAMP-1 and VAMP-2; Torpedo VAMP-1; Aplysia VAMP;squid VAMP; Drosophila syb and n-syb; and leech VAMP. Thus, a BoNT/Drecognition sequence can correspond, for example, to a segment of humanVAMP-1 or VAMP-2, mouse VAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2, ratVAMP-1 or VAMP-2, rat cellubrevin, chicken VAMP-1 or VAMP-2, TorpedoVAMP-1, Aplysia VAMP, squid VAMP, Drosophila syb or n-syb, leech VAMP,or another naturally occurring protein sensitive to cleavage by BoNT/D.Furthermore, as shown in Table D above, comparison of native VAMP aminoacid sequences cleaved by BoNT/D reveals significant sequencevariability (see, also, FIG. 4), indicating that a variety of amino acidsubstitutions and modifications relative to a naturally occurringBoNT/D-sensitive VAMP sequence can be tolerated in a BoNT/D substrateuseful in the invention.

As used herein, the term “botulinum toxin serotype E recognitionsequence” is synonymous with “BoNT/E recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements, or both, sufficient for detectable proteolysis at the scissilebond by a BoNT/E under appropriate conditions. A scissile bond cleavedby BoNT/E can be, for example, Arg-Ile.

One skilled in the art appreciates that a BoNT/E recognition sequencecan correspond to a segment of a protein that is sensitive to cleavageby botulinum toxin serotype E, or can be substantially similar to asegment of a BoNT/E-sensitive protein. A variety of naturally occurringproteins sensitive to cleavage by BoNT/E are known in the art andinclude, for example, human, mouse and rat SNAP-25; mouse SNAP-23;chicken SNAP-25; goldfish SNAP-25A and SNAP-25B; zebrafish SNAP-25; C.elegans SNAP-25; and leech SNAP-25 (see Table B). Thus, a BoNT/Erecognition sequence can correspond, for example, to a segment of humanSNAP-25, mouse SNAP-25, rat SNAP-25, mouse SNAP-23, chicken SNAP-25,goldfish SNAP-25A or 25B, C. elegans SNAP-25, leech SNAP-25, or anothernaturally occurring protein sensitive to cleavage by BoNT/E.Furthermore, as shown in Table B and FIG. 3 above, comparison of nativeSNAP-23 and SNAP-25 amino acid sequences cleaved by BoNT/E reveals thatsuch sequences are not absolutely conserved, indicating that a varietyof amino acid substitutions and modifications relative to a naturallyoccurring BoNT/E-sensitive SNAP-23 or SNAP-25 sequence can be toleratedin a BoNT/E substrate useful in the invention.

The term “botulinum toxin serotype F recognition sequence,” as usedherein, is synonymous with “BoNT/F recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements, or both, sufficient for detectable proteolysis at the scissilebond by a BoNT/F under appropriate conditions. A scissile bond cleavedby BoNT/F can be, for example, Gln-Lys.

A variety of BoNT/F recognition sequences are well known in the art orcan be defined by routine methods. A BoNT/F recognition sequence caninclude, for example, residues 27 to 116; residues 37 to 116; residues 1to 86; residues 1 to 76; or residues 1 to 69 of rat VAMP-2 (SEQ ID NO:90; Yamasaki et al., supra, 1994). A BoNT/F recognition sequence alsocan include, for example, residues 27 to 69 or residues 37 to 69 of ratVAMP-2 (SEQ ID NO: 90). It is understood that a similar BoNT/Frecognition sequence can be prepared, if desired, from a corresponding(homologous) segment of another BoNT/F-sensitive VAMP isoform or homologsuch as human VAMP-1 or human VAMP-2.

A BoNT/F recognition sequence can correspond to a segment of a proteinthat is sensitive to cleavage by botulinum toxin serotype F, or can besubstantially similar to a segment of a BoNT/F-sensitive protein. Avariety of naturally occurring proteins sensitive to cleavage by BoNT/Fare known in the art and include, for example, human, mouse and bovineVAMP-1 and VAMP-2; rat VAMP-1 and VAMP-2; rat cellubrevin; chickenVAMP-1 and VAMP-2; Torpedo VAMP-1; Aplysia VAMP; Drosophila syb; andleech VAMP (see Table D). Thus, a BoNT/F recognition sequence cancorrespond, for example, to a segment of human VAMP-1 or VAMP-2, mouseVAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2, rat VAMP-1 or VAMP-2, ratcellubrevin, chicken VAMP-1 or VAMP-2, Torpedo VAMP-1, Aplysia VAMP,Drosophila syb, leech VAMP, or another naturally occurring proteinsensitive to cleavage by BoNT/F. Furthermore, as shown in Table D above,comparison of native VAMP amino acid sequences cleaved by BoNT/F revealsthat such sequences are not absolutely conserved (see, also, FIG. 4),indicating that a variety of amino acid substitutions and modificationsrelative to a naturally occurring BoNT/F-sensitive VAMP sequence can betolerated in a BoNT/F substrate useful in the invention.

As used herein, the term “botulinum toxin serotype G recognitionsequence” is synonymous with “BoNT/G recognition sequence” and means ascissile bond together with adjacent or non-adjacent recognitionelements, or both, sufficient for detectable proteolysis at the scissilebond by a BoNT/G under appropriate conditions. A scissile bond cleavedby BoNT/G can be, for example, Ala-Ala.

A BoNT/G recognition sequence can correspond to a segment of a proteinthat is sensitive to cleavage by botulinum toxin serotype G, or can besubstantially similar to such a BoNT/G-sensitive segment. As illustratedin Table D above, a variety of naturally occurring proteins sensitive tocleavage by BoNT/G are known in the art and include, for example, human,mouse and bovine VAMP-1 and VAMP-2; rat VAMP-1 and VAMP-2; ratcellubrevin; chicken VAMP-1 and VAMP-2; and Torpedo VAMP-1. Thus, aBoNT/G recognition sequence can correspond, for example, to a segment ofhuman VAMP-1 or VAMP-2, mouse VAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2,rat VAMP-1 or VAMP-2, rat cellubrevin, chicken VAMP-1 or VAMP-2, TorpedoVAMP-1, or another naturally occurring protein sensitive to cleavage byBoNT/G. Furthermore, as shown in Table D above, comparison of nativeVAMP amino acid sequences cleaved by BoNT/G reveals that such sequencesare not absolutely conserved (see, also, FIG. 4), indicating that avariety of amino acid substitutions and modifications relative to anaturally occurring BoNT/G-sensitive VAMP sequence can be tolerated in aBoNT/G substrate useful in the invention.

As used herein, the term “tetanus toxin recognition sequence” means ascissile bond together with adjacent or non-adjacent recognitionelements, or both, sufficient for detectable proteolysis at the scissilebond by a tetanus toxin under appropriate conditions. A scissile bondcleaved by TeNT can be, for example, Gln-Phe.

A variety of TeNT recognition sequences are well known in the art or canbe defined by routine methods and include sequences corresponding tosome or all of the hydrophilic core of a VAMP protein such as humanVAMP-1 or human VAMP-2. A TeNT recognition sequence can include, forexample, residues 25 to 93 or residues 33 to 94 of human VAMP-2 (SEQ IDNO: 8; Cornille et al., Eur. J. Biochem. 222:173-181 (1994); Foran etal., Biochem. 33: 15365-15374 (1994)); residues 51 to 93 or residues 1to 86 of rat VAMP-2 (SEQ ID NO: 90; Yamasaki et al., supra, 1994); orresidues 33 to 94 of human VAMP-1 (SEQ ID NO: 7). A TeNT recognitionsequence also can include, for example, residues 25 to 86, residues 33to 86 or residues 51 to 86 of human VAMP-2 (SEQ ID NO: 8) or rat VAMP-2(SEQ ID NO: 90). It is understood that a similar TeNT recognitionsequence can be prepared, if desired, from a corresponding (homologous)segment of another TeNT-sensitive VAMP isoform or species homolog suchas human VAMP-1 or sea urchin or Aplysia VAMP.

Thus, a TeNT recognition sequence can correspond to a segment of aprotein that is sensitive to cleavage by tetanus toxin, or can besubstantially similar to a segment of a TeNT-sensitive protein. As shownin Table D above, a variety of naturally occurring proteins sensitive tocleavage by TeNT are known in the art and include, for example, human,mouse and bovine VAMP-1 and VAMP-2; rat VAMP-2; rat cellubrevin; chickenVAMP-2; Torpedo VAMP-1; sea urchin VAMP; Aplysia VAMP; squid VAMP; C.elegans VAMP; Drosophila n-syb; and leech VAMP. Thus, a TeNT recognitionsequence can correspond, for example, to a segment of human VAMP-1 orVAMP-2, mouse VAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2, rat VAMP-2, ratcellubrevin, chicken VAMP-2, Torpedo VAMP-1, sea urchin VAMP, AplysiaVAMP, squid VAMP, C. elegans VAMP, Drosophila n-syb, leech VAMP, oranother naturally occurring protein sensitive to cleavage by TeNT.Furthermore, comparison of native VAMP amino acid sequences cleaved byTeNT reveals that such sequences are not absolutely conserved (Table Dand FIG. 4). This finding indicates that a variety of amino acidsubstitutions and modifications relative to a naturally occurringTeNT-sensitive VAMP sequence can be tolerated in a TeNT substrate usefulin the invention.

As used herein, the term “peptidomimetic” is used broadly to mean apeptide-like molecule that is cleaved by the same clostridial toxin asthe peptide substrate upon which it is structurally based. Suchpeptidomimetics include chemically modified peptides, peptide-likemolecules containing non-naturally occurring amino acids, and peptoids,which are peptide-like molecules resulting from oligomeric assembly ofN-substituted glycines, and are cleaved by the same clostridial toxin asthe peptide substrate upon which the peptidomimetic is derived (see, forexample, Goodman and Ro, Peptidomimetics for Drug Design, in “Burger'sMedicinal Chemistry and Drug Discovery” Vol. 1 (ed. M. E. Wolff; JohnWiley & Sons 1995), pages 803-861).

A variety of peptidomimetics are known in the art including, forexample, peptide-like molecules which contain a constrained amino acid,a non-peptide component that mimics peptide secondary structure, or anamide bond isostere. A peptidomimetic that contains a constrained,non-naturally occurring amino acid can include, for example, anα-methylated amino acid; an α,α-dialkyl-glycine or α-aminocycloalkanecarboxylic acid; an N^(α)-C^(α) cyclized amino acid; an N-methylatedamino acid; β- or γ-amino cycloalkane carboxylic acid; anα,β-unsaturated amino acid; a β,β-dimethyl or β-methyl amino acid; aβ-substituted-2,3-methano amino acid; an NC^(δ) or C^(α)-C^(δ) cyclizedamino acid; or a substituted proline or another amino acid mimetic. Inaddition, a peptidomimetic which mimics peptide secondary structure cancontain, for example, a nonpeptidic β-turn mimic; γ-turn mimic; mimic ofβ-sheet structure; or mimic of helical structure, each of which is wellknown in the art. A peptidomimetic also can be a peptide-like moleculewhich contains, for example, an amide bond isostere such as aretro-inverso modification; reduced amide bond; methylenethioether ormethylenesulfoxide bond; methylene ether bond; ethylene bond; thioamidebond; trans-olefin or fluoroolefin bond; 1,5-disubstituted tetrazolering; ketomethylene or fluoroketomethylene bond or another amideisostere. One skilled in the art understands that these and otherpeptidomimetics are encompassed within the meaning of the term“peptidomimetic” as used herein.

In any of the methods of the invention, a clostridial toxin substratecan include one or multiple clostridial toxin cleavage sites for thesame or different clostridial toxins. In particular embodiments, theinvention provides methods that rely on a clostridial toxin substratewhich contains a single clostridial toxin cleavage site. In otherembodiments, the invention provides methods which rely on a clostridialtoxin substrate which contains multiple cleavage sites for the sameclostridial toxin. These cleavage sites can be incorporated within thesame or different clostridial toxin recognition sequences. Asnon-limiting examples, a clostridial toxin substrate can have multiplecleavage sites for the same clostridial toxin intervening between thesame fluorophore and bulking group or the same donor fluorophore andacceptor. A clostridial toxin substrate useful in the invention cancontain, for example, two or more, three or more, five or more, or tenor more cleavage sites for the same clostridial toxin. A clostridialtoxin substrate useful in the invention also can have, for example, two,three, four, five, six, seven, eight, nine or ten cleavage sites for thesame clostridial toxin; the multiple cleavage sites can intervenebetween the same or different fluorophores and bulking groups, orbetween the same or different donor fluorophores and acceptors.

A clostridial toxin substrate useful in the invention also can includecleavage sites for different clostridial toxins. In particularembodiments, the invention provides a method that relies on aclostridial toxin substrate which includes multiple cleavage sites fordifferent clostridial toxins all intervening between the samefluorophore and bulking group, or between the same donor fluorophore andacceptor. A clostridial toxin substrate can include, for example,cleavage sites for two or more, three or more, or five or more differentclostridial toxins all intervening between the same fluorophore andbulking group. A clostridial toxin substrate also an include, forexample, cleavage sites for two or more, three or more, or five or moredifferent clostridial toxins all intervening between the same donorfluorophore and acceptor. A clostridial toxin substrate also canincorporate, for example, cleavage sites for two or more, three or more,or five or more different clostridial toxins intervening between atleast two fluorophore-bulking group pairs or between at least two donorfluorophore-acceptor pairs. In particular embodiments, the inventionprovides a clostridial toxin substrate having cleavage sites for two,three, four, five, six, seven or eight different clostridial toxins,where the cleavage sites intervene between the same or differentfluorophores and bulking groups, or between the same or different donorfluorophores and acceptors. In further embodiments, the inventionprovides a clostridial toxin substrate which has any combination of two,three, four, five, six, seven or eight cleavage sites for anycombination of the following clostridial toxins: BoNT/A, BoNT/B,BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G and TeNT.

A method of the invention optionally can be performed with multiplesubstrates. In a method of the invention which relies on a clostridialtoxin substrate containing a donor fluorophore-acceptor pair, aclostridial toxin substrate is treated with a sample, the substrateincluding a first donor fluorophore, a first acceptor having anabsorbance spectrum which overlaps the emission spectrum of the firstdonor fluorophore, and a first clostridial toxin recognition sequencecontaining a cleavage site, where the cleavage site intervenes betweenthe donor fluorophore and the acceptor and where, under the appropriateconditions, resonance energy transfer is exhibited between the firstdonor fluorophore and the first acceptor. If desired, a secondclostridial toxin substrate can be included in the same assay; thissecond substrate contains a second donor fluorophore and second acceptorhaving an absorbance spectrum which overlaps the emission spectrum ofthe second donor fluorophore, and a second clostridial toxin recognitionsequence that is cleaved by a different clostridial toxin than the toxinthat cleaves the first clostridial toxin recognition sequence. The donorfluorophore-acceptor pair in the second substrate can be the same ordifferent from the donor fluorophore-acceptor pair in the firstsubstrate. In this way, a single sample can be simultaneously assayedfor the presence of more than one clostridial toxin.

In a method of the invention which relies on a clostridial toxinsubstrate containing a donor fluorophore-acceptor pair, it is understoodthat one can assay for any combination of clostridial toxins, forexample, two, three, four, five, six, seven, eight, or more clostridialtoxins. One can assay, for example, any combination of two, three, four,five, six, seven or eight of BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E,BoNT/F, BoNT/G and TeNT. As an example, an assay can be performed withseven substrates, each of which includes fluorescein andtetramethylrhodamine flanking a BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E,BoNT/F or BoNT/G recognition sequence and cleavage site. Thesesubstrates can be treated with a sample under conditions suitable forbotulinum toxin activity before exciting the donor fluorescein withplane polarized light at an absorption wavelength of about 488 nm anddetermining fluorescence polarization. A change in the fluorescencepolarization is indicative of the presence or activity of at least oneclostridial toxin. Such an assay can be useful, for example, forassaying food samples or tissue samples for the presence of anybotulinum or other clostridial toxin and can be combined, if desired,with one or more subsequent assays for individual clostridial toxins orspecific combinations of clostridial toxins.

In another embodiment, a single sample is assayed for two or moredifferent clostridial toxins using two or more different clostridialtoxin substrates, with each substrate containing a different donorfluorophore-acceptor pair. The use of multiple substrates can be usefulfor extending the dynamic range of an assay, as described, for example,in U.S. Pat. No. 6,180,340. As an example of the use of multipleclostridial toxin substrates, a single sample can be assayed for thepresence or activity of BoNT/A and BoNT/B using first and secondclostridial toxin substrates: the first clostridial toxin substratecontains the donor fluorophore Alexa Fluor® 555 and the acceptor AlexaFluor® 568 with an intervening BoNT/A recognition sequence, and a secondclostridial toxin substrate contains the donor fluorophore Alexa Fluor®700 and the acceptor Alexa Fluor® 750 with an intervening BoNT/Brecognition sequence. Those skilled in the art understand that the firstdonor fluorophore can be excited before or after excitation of thesecond donor fluorophore, and that the change in fluorescencepolarization of the first substrate can be determined before, at thesame time, or after determining energy transfer of the second substrate.

In a further embodiment, a method of the invention is useful forassaying two or more different purified or isolated clostridial toxinsusing two or more different clostridial toxin substrates, with eachsubstrate containing the same donor fluorophore-acceptor pair. In theendpoint format, the presence or activity of different serotypes isassayed by adding the serotypes sequentially and waiting betweenadditions for the response to stabilize.

EXAMPLES

The following examples are intended to illustrate but not limit thepresent invention.

Example I Preparation gf GFP-Snap25-His6-C-Alexa Fluor® 594 andGFP-Snap25-His6-C-Alexa Fluor®546 Substrates

This example describes construction of substrates suitable for assayingfor the presence or activity of a clostridial toxin using fluorescencepolarization.

A. Construction of GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C

A substrate was prepared as a fusion protein containing greenfluorescent protein (GFP), murine SNAP-25 residues 134-206, apolyhistidine affinity tag (6×His), and a carboxy-terminal cysteine,with several components separated by peptide linkers. As describedfurther below, the substrate was designed such that the GFP and terminalcysteine were present at opposite ends of SNAP-25₍₁₃₄₋₂₀₆₎.

The SNAP-25 sequence was obtained from pT25FL, a plasmid which containsthe full-length mouse SNAP-25 gene inserted in frame with the 3′terminus of the glutathione-S-transferase (GST) gene(GST-SNAP25₍₁₋₂₀₆₎), provided by Professor Dolly (O'Sullivan et al., J.Biol. Chem. 274:36897-36904 (1999)). The SNAP-25 sequence from pT25FLwas incorporated into a second expression vector, which was designed tohave a BirAsp signal sequence for biotinylation and a polyhistidineaffinity tag fused to the amino-terminus of residues 134 to 206 ofSNAP-25 (BirAsp-polyHis-SNAP25₍₁₃₄₋₂₀₆₎, denoted “BA-SNAP”). The DNAsequence encoding SNAP25₍₁₃₄₋₂₀₆₎ was generated by PCR amplification ofthe appropriate region of the pT25FL plasmid with PCR primers 5′-GCT AGATCT CGA GTT AAC CAC TTC CCA GCA TCT TTG-3′ (SEQ ID NO: 91; antisense)and 5′-ATC CGG AGG GTA ACA AAC GAT GCC-3′ (SEQ ID NO: 92; sense) toproduce a SNAP25₍₁₃₄₋₂₀₆₎ PCR product containing a Bgl II restrictionsite (PCR product A).

The BirAsp sequence, a natural substrate for biotinylation, as well as apolyhistidine affinity tag, were engineered for fusion upstream and inframe with the SNAP25₍₁₃₄₋₂₀₆₎ sequence using synthetic oligonucleotidesSEQ ID NOS: 93 and 94, which contained a 20 bp complementary region.These oligonucleotides, 5′-CGA ATT CCG CGG GCC ACC ATG GGA GGA GGA CTGAAC GAC ATC TTC GAG GCT CAA AAG ATC-3′ (SEQ ID NO: 93; sense; Sac IIsite underlined) and 5′-TCG TTT GTT ACC CTC CGG ATA TGA TGA TGA TGA TGATGA TGA TGG GAT CCA TGC CAC TCG ATC TTT TGA GCC TCG AAG A-3′ (SEQ ID NO:94; antisense), were annealed, and the single strand overhangs filled byPCR amplification to yield PCR product B.

The two double stranded PCR products containing the coding sequences forSNAP25₍₁₃₄₋₂₀₆₎, denoted PCR product A, and BirAsp and polyhistidine,denoted PCR product B, were denatured and annealed. The 20 bpcomplementary sequence in the two gene fragments is shown in italics inPCR primers SEQ ID NO: 92 and SEQ ID NO: 94). After filling in theoverhangs by PCR, the product was amplified with primers SEQ ID NO: 93and SEQ ID NO: 91. The resulting PCR product, which encodedBirAsp-polyHis-SNAP25₍₁₃₄₋₂₀₆₎ (designated “BA-SNAP”), was digested withSacII and BglII, and the isolated gene insert ligated into pQBI25fA2vector digested with SacII and BamHI, to yield plasmid pNTP12 (pQBI25fA2containing BA-SNAP).

For expression and purification from E. coli, the BA-SNAP gene wastransferred into a pTrc99A plasmid (Amersham Pharmacia Biotech). TheBA-SNAP gene was isolated from pNTP12 by digestion with NcoI and XhoIfollowed by gel purification. Separately, the pTrc99A plasmid wasdigested with NcoI and SalI, and the isolated vector ligated to theBA-SNAP gene to yield plasmid pNTP14 (pTrc99A containing BA-SNAP).

For cloning of the BA-SNAP gene into plasmid pQE-50, the BA-SNAPfragment was PCR amplified from pNTP14 with primer SEQ ID NO: 91 andprimer SEQ ID NO: 95 (5′-CGA AGA TCT GGA GGA CTG AAC GAC ATC TTC-3′(sense; Bgl II site underlined)). After digestion with BglII and XhoI,the amplified PCR product was ligated into vector pQE-50, which had beendigested with BamH I and Sal I. The resulting plasmid, which representspQE50 containing BA-SNAP, was designated pNTP26.

A plasmid encoding the green fluorescent protein (GFP) fusion proteinsubstrate was prepared by modifying vector pQBI T7-GFP (QuantumBiotechnologies; Carlsbad, Calif.) in three phases as described below.First, vector pQBI T7-GFP was PCR-modified to remove the stop codon atthe 3′ terminus of the GFP-coding sequence and to insert the codingsequence for a portion of the peptide linker separating GFP from theSNAP-25 fragment. Second, a DNA fragment coding for SNAP-25₍₁₃₄₋₂₀₆₎ wasPCR amplified from pNTP26 using PCR primers designed to incorporate thecoding sequence for the remainder of the peptide linker fused 5′ to theSNAP-25₍₁₃₄₋₂₀₆₎ gene and a 6×His affinity tag fused 3′ of the gene. Theresultant PCR product was cloned into the modified pQBI vector describedabove to yield pQBI GFP-SNAP25₍₁₃₄₋₂₀₆₎.

Plasmid pQBI GFP-SNAP25₍₁₃₄₋₂₀₆₎ was then modified by site-directedmutagenesis to add a cysteine codon at the carboxy-terminus using primerSEQ ID NO: 96 (5′-GATGGTGATGGTGATGACAGCCGCCACC GCCACC-3′ (antisenseprimer, with the added nucleotides underlined) and its reversecomplement (sense primer). The resulting plasmid, designated pQBIGFP-SNAP25 (Cys-Stop), is shown in FIG. 6A and was used for expressionof GFP-SNAP25₍₁₃₄₋₂₀₆₎-6×His-Cys. The nucleic acid and predicted aminoacid sequence for the GFP-SNAP25₍₁₃₄₋₂₀₆₎-6×His-cysteine construct isshown herein in FIG. 6B.

B. Expression and Characterization of GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C

The pQBI GFP-SNAP25 (Cys-Stop) expression vector was transformed into E.coli BL21(DE3) cells (Novagen; Madison, Wis.; or Invitrogen; Carlsbad,Calif.) or into E. coli BL21-CodonPlus®(DE3)-RIL cells (Stratagene)containing the T7 RNA polymerase gene. Transformed cells were selectedon LB-ampicillin plates overnight at 37° C. Single colonies were used toinoculate 1-3 mL starter cultures, which were in turn used to inoculate0.5 to 1.0 L cultures. The large cultures were grown at 37° C. withshaking until A₅₉₅ reached 0.5-0.6, at which time they were removed fromthe incubator and were allowed to cool briefly. After induction ofprotein expression with 1 mM IPTG, GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C substratewas expressed from the pQBI GFP-SNAP25 (Cys-Stop) plasmid overnight withshaking at 16° C. in order to facilitate formation of the GFPfluorophore. Cells from 250 mL aliquots of the expression cultures werecollected by centrifugation (30 minutes, 6,000×g, 4° C.) and stored at−80° C. until needed.

Substrates were purified at 4° C. by a two-step procedure involving IMACpurification, followed by a de-salting step to remove NaCl andimidazole, typically yielding greater than 150 mg/L of purifiedsubstrate as follows. Cell pellets from 250 mL cultures were eachresuspended in 7-12 mL Column Binding Buffer (25 mM HEPES, pH 8.0; 500mM NaCl; 1 mM β-mercaptoethanol; 10 mM imidazole), lysed by sonication(1 minute 40 seconds in 10-second pulses at 38% amplitude), andclarified by centrifugation (16000 rpm, 4° C., 1 hour). Affinity resin(3-5 mL Talon SuperFlow Co²⁺ per cell pellet) was equilibrated in aglass or disposable column support (Bio-Rad) by rinsing with 4 columnvolumes of sterile ddH₂O and 4 column volumes of Column Binding Buffer.Clarified lysate was applied to the column in one of two ways: (1)Lysate was added to the resin and batch bound by horizontal incubationfor 1 hour with gentle rocking or (2) Lysate was applied to the verticalcolumn and allowed to enter the column slowly by gravity flow. Followingbatch binding only, the column was righted and the solution drained,collected, and passed over the resin again. In both cases, after thelysate had been applied, the column was washed with 4-5 column volumesof Column Binding Buffer. In some cases, the column was further washedwith 1-2 column volumes of Column Wash Buffer (25 mM HEPES, pH 8.0; 500mM NaCl; 1 mM β-mercaptoethanol; 20 mM imidazole). Protein was elutedwith 1.5 to 2.0 column volumes of Column Elution Buffer (25 mM HEPES, pH8.0; 500 mM NaCl; 1 mM β-mercaptoethanol; 250 mM imidazole), which wascollected in fractions of ˜1.4 mL. The green fractions were combined,concentrated with a centrifugal filter (10,000 or 30,000 molecularweight cut-off) and desalted by FPLC (BioRad Biologic DuoLogic, QuadTecUV-Vis detector) with a HiPrep 26/10 size exclusion column (Pharmacia)and an isocratic mobile phase of chilled Fusion Protein Desalting Buffer(50 mM HEPES, pH 7.4, 4° C.) at a flow rate of 10 mL/minute. Desaltedprotein was collected as a single fraction, and the concentrationdetermined using a BioRad Protein Assay. The GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-Csubstrate was analyzed by reducing SDS-PAGE. The protein solution wassubsequently divided into 500 μL aliquots, flash-frozen with liquidnitrogen and stored at −80° C. Once defrosted, a working aliquot wasstored at 4° C., protected from light.

C. Labeling with Alexa Fluor® 594 and Alexa Fluor® 546

The GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C construct contains a single cysteineresidue which is solvent exposed although there are three buriedcysteine residues within GFP which are not available for chemicalmodification (Selvin, supra, 2000; Heyduk, Curr. Opin. Biotech.13:292-296 (2002)). The carboxy-terminal cysteine residue can thereforebe selectively labeled using a fluorophore-maleimide at neutral pH.Shown in FIGS. 7A and 7B, respectively, are the absorption andemission/excitation spectra of purified GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-Cprotein. The concentration of the protein solution was determined to be2.74 mg/ml based on the theoretical molar extinction coefficient of20250 M⁻¹cm⁻¹ as calculated from the primary sequence of the construct.The molecular weight of the purified GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C proteinwas confirmed to be about 37,000 using Matrix Assisted Laser DesorptionTime of Flight mass spectrometry (MALDI-TOF).

Labeling with Alexa Fluor® 594 was performed essentially as follows. TheC-terminal cysteine residue of the GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C proteinwas labeled by adding a concentrated solution of Alexa Fluor® 594(Molecular Probes, Inc.) in dry dimethyl formamide (DMF) to a finalconcentration of 20:1 molar excess of fluorophore to protein. Theprotein/fluorophore solution was kept at 4° C. in the refrigeratorovernight and subsequently dialyzed against 20 mM HEPES pH 6.9.

The absorption spectrum of the GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C proteinlabeled with Alexa Fluor® 594 is shown in FIG. 8A following dialysisagainst 20 mM HEPES pH 6.9, which is the pH used for assaying enzymaticactivity of reduced bulk toxin or purified BoNT-A light chain. Thelabeling ratio, as calculated from the absorption spectrum using thetheoretical extinction coefficient of the GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-Cconstruct, was approximately 3:1 (protein: Alexa probe). Shown in FIG.8B are the excitation and emission spectra of labeledGFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C-Alexa Fluor® 594 after extensive dialysis for20 hours with three changes of buffer to remove free probe.

Labeling with Alexa Fluor® 546 was performed essentially as follows withall procedures carried out on ice or at 4° C. Four microliters of a 10mM aqueous solution of Alexa Fluor® 546 C₅ maleimide (MW 1,034.37;Molecular Probes) were added to 200 μL of GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C(135 μM in 25 mM HEPES buffer, pH 7.2), mixed well, and incubated at 4°C. overnight. The reactions were transferred to Biomax Ultrafreecentrifugal filters (30 KDa NMWL; Millipore), concentrated, and thenreconcentrated two times from 25 mM HEPES, pH 7.2, to remove most of theexcess Alexa Fluor® 546. To remove the remaining unreacted Alexa Fluor®546, the concentrated solutions were transferred to Spin Microdialyzers(Harvard Apparatus) and each was dialyzed against 500 mL 20 mM HEPES, pH6.9, for 1 hour, and against 3×250 mL of that buffer for about 1.5 hourseach. A small aliquot was removed for fluorescence measurements, and thebalance of the reaction was flash-frozen in liquid nitrogen and storedat −80° C.

Example II Clostridial Toxin Complex Activity Assayed Using FluorescencePolarization

This example demonstrates that a fluorescence polarization assay can beused to determine the presence or activity of a clostridial toxin.

The GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C protein labeled with Alexa Fluor® 594 wastested for its utility as a suitable substrate for BoNT/A reduced bulktoxin by recording the change in polarization over time. A Cary Eclipsespectrofluorometer (Varian, Inc.; Palo Alto, Calif.) equipped withmotorized thin-film polarizers was used to monitor the reaction. Theexcitation wavelength was set at 590 nm, and polarized emission wasrecorded at 620 nm.

Several dilutions of BoNT/A bulk toxin or BoNT/A light chain wereprepared, and fluorescence polarization monitored over time. For bothbulk toxin and light chain, fluorescence polarization was reduced at orshortly after the time the diluted toxin was added. FIG. 9 shows thedata for bulk BoNT/A toxin proteolysis ofGFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C-Alexa Fluor® 594. As shown in panel 9D, toxinwas detected at a concentration of as little as about 50 ng/ml.

These results demonstrate that the presence or activity of a clostridialtoxin can be sensitively determined using synthetic substrates assayedby fluorescence polarization.

Example III Clostridial Toxin Complex Activity Assayed UsingFluorescence Polarization in Combination with Fluorescence ResonanceEnergy Transfer

This example demonstrates that fluorescence polarization can be assayedto determine the presence or activity of a clostridial toxin using asubstrate which exhibits fluorescence resonance energy transfer.

The GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C protein labeled with Alexa Fluor® 546 asdescribed above was utilized as a substrate for BoNT/A. As indicatedabove, the photoselection properties of GFP and Alexa Fluor® 546 providefor fluorescence resonance energy transfer (FRET) between the donorfluorophore GFP and the acceptor Alexa Fluor® 546. Steady-statepolarization measurements were carried out in a Cary Eclipsespectrophotometer (Varian). Excitation was at 474 nm, the excitationmaximum of the GFP component. Emission was measured at the Alexa Fluor®546 fluorescence maximum of 570 nm. In all cases, a dual path lengthcuvette (10 mm by 2 mm) was utilized, and the emission viewed throughthe 2 mm path. A solution of 390 μL Toxin Reaction Buffer (50 mM HEPES,pH 7.2; 0.1% v/v TWEEN-20; 10 μM Zn Cl₂, 10 mM DTT) and 10 μL ofGFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C-Alexa Fluor® 546 was placed in the cuvetteand allowed to equilibrate to 30° C. When the polarization measurements,which were taken at 30 second intervals, were stabilized, 10 μL ofrecombinant BoNT/A light chain (rLC/A) at a concentration of 1.0 μg/μL,0.5 μg/μL, 0.25 μg/μL, or 0.1 μg/μL was added to the cuvette.Measurements continued to be taken until the polarization againstabilized.

As shown in FIG. 10, fluorescence polarization increased upon additionof recombinant BoNT/A light chain which results in substrate cleavage.As compared to the substrate having GFP and Alexa Fluor® 594, thefluorescence resonance energy transfer enhanced the polarization changeupon turnover, thereby increasing the sensitivity of the assay. Theoverall change in polarization using theGFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C-Alexa Fluor® 546 substrate was about 40 mP,twice the magnitude of the depolarization of approximately 20 mPobserved during proteolysis of GFP-SNAP25₍₁₃₄₋₂₀₆₎-His6-C-Alexa Fluor®594.

These results indicate that fluorescence polarization can be combinedwith fluorescence resonance energy transfer for enhanced sensitivity inassaying for the presence or activity of a clostridial toxin.

All journal article, reference and patent citations provided above, inparentheses or otherwise, whether previously stated or not, areincorporated herein by reference in their entirety.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1. A method of determining the presence or activity of a clostridialtoxin, comprising the steps of: (a) treating with a sample, including aclostridial toxin, under conditions suitable for clostridial toxinprotease activity, a clostridial toxin substrate, said clostridial toxinsubstrate comprising (i) a donor fluorophore; (ii) an acceptor having anabsorbance spectrum overlapping the emission spectrum of said donorfluorophore; and (iii) a clostridial toxin recognition sequencecomprising a cleavage site, wherein said cleavage site intervenesbetween said donor fluorophore and said acceptor; and (b) measuringfluorescence polarization of said treated clostridial toxin substrate byexciting said fluorophore with plane polarized light; wherein anincrease in said fluorescence polarization is indicative of the activityof said clostridial toxin in said sample due to cleavage of said treatedclostridial toxin substrate by said clostridial toxin; and wherein adecrease in said fluorescence polarization is indicative of the presenceof said clostridial toxin in said sample due to complex formation ofsaid clostridial toxin with said treated clostridial toxin substrate. 2.The method of claim 1, wherein said donor fluorophore is a fluorescentprotein.
 3. The method of claim 2, wherein said fluorescent protein is agreen fluorescent protein, a blue fluorescent protein, a cyanfluorescent protein, a yellow fluorescent protein, or a red fluorescentprotein .
 4. The method of claim 1, wherein said donor fluorophore is afluorescein arsenical hairpin binding dye or a resorufin arsenicalhairpin binding dye.
 5. The method of claim 1, wherein said donorfluorophore is selected from a fluorescin or a fluorescin derivative, arhodamine or a rhodamine derivative, and a cyanine or a cyaninederivative.
 6. The method of claim 5, wherein said fluorescin or saidfluorescin derivative is selected from diaminotriazinylamino-fluorescein(DTAF), carboxyfluorescein (FAM), a biarsenic-based fluorescein orfluorescein derivative, and a dipyrromethene boron difluoride-basedfluorescein or fluorescein derivative.
 7. The method of claim 5, whereinsaid rhodamine or said rhodamine derivative is selected fromtetramethylcarboxyrhodamine (TMR), carboxy-x-rhodamine (ROX), rhodaminegreen, octadecylrhodamine, a biarsenic-based rhodamine or rhodaminederivative, and a dipyrromethene boron difluoride-based rhodamine orrhodamine derivative.
 8. The method of claim 5, wherein said cyanine orsaid cyanine derivative is indocarbocyanine.
 9. The method of claim 1,wherein said donor fluorophore has a fluorescence lifetime of at least0.5 nanoseconds, at least 5 nanoseconds, or at least 10 nanoseconds. 10.The method of claim 1, wherein said acceptor is a fluorescent protein.11. The method of claim 10, wherein said fluorescent protein is a greenfluorescent protein, a blue fluorescent protein, a cyan fluorescentprotein, a yellow fluorescent protein, or a red fluorescent protein .12. The method of claim 1, wherein said acceptor is a fluoresceinarsenical hairpin binding dye or a resorufin arsenical hairpin bindingdye.
 13. The method of claim 1, wherein said acceptor is selected from afluorescin or a fluorescin derivative, a rhodamine or a rhodaminederivative, and a cyanine or a cyanine derivative.
 14. The method ofclaim 13, wherein said fluorescin or said fluorescin derivative isselected from diaminotriazinylamino-fluorescein (DTAF),carboxyfluorescein (FAM), a biarsenic-based fluorescein or fluoresceinderivative, and a dipyrromethene boron difluoride-based fluorescein orfluorescein derivative.
 15. The method of claim 13, wherein saidrhodamine or said rhodamine derivative is selected fromtetramethylcarboxyrhodamine (TMR), carboxy-x-rhodamine (ROX), rhodaminegreen, octadecylrhodamine, a biarsenic-based rhodamine or rhodaminederivative, and a dipyrromethene boron difluoride-based rhodamine orrhodamine derivative.
 16. The method of claim 13, wherein said cyanineor said cyanine derivative is indocarbocyanine.
 17. The method of claim1, wherein said acceptor has a fluorescence lifetime of at least 0.5nanoseconds, at least 5 nanoseconds, or at least 10 nanoseconds.
 18. Themethod of claim 1, wherein said clostridial toxin recognition sequencecomprises at least 100 residues or at least 200 residues.
 19. The methodof claim 1, wherein said clostridial toxin recognition sequence is aBoNT/A recognition sequence, a BoNT/B recognition sequence, a BoNT/C1recognition sequence, a BoNT/D recognition sequence, a BoNT/Erecognition sequence, a BoNT/F recognition sequence, a BoNT/Grecognition sequence, or a TeNT recognition sequence.
 20. The method ofclaim 1, wherein said clostridial toxin substrate further comprises abulking group.
 21. The method of claim 1, wherein said bulking group isgreater than 1 KDa, greater than 5 kDa, or greater than 10 kDa
 22. Themethod of claim 1, wherein in step (b) said increased fluorescencepolarization or said decreased fluorescence polarization at least 5 mP.23. The method of claim 1, wherein in step (e) step (b) said increasedfluorescence polarization or said decreased fluorescence polarization atleast 15 mP.
 24. The method of claim 1, wherein said sample is a cell, acrude cell lysate, or a fractionated or partially purified cell lysate.25. The method of claim 1, wherein said clostridial toxin is an isolatedclostridial toxin or an isolated clostridial toxin light chain.
 26. Themethod of claim 1, wherein said clostridial toxin is a formulatedclostridial toxin product.